Lab on a Chip

Given the emergence of antimicrobial drug resistance, it is critical to understand the heterogeneity of response to an antibiotic within a population of cells. Since the drug can exert a selection pressure that leads to the emergence of resistant phenotypes. To date, neither bulk nor single-cell methods are able to link the heterogeneity of single-cell susceptibility to the population-scale response to antibiotics. Here we present a platform that measures the ability of individual E. coli cells to form small colonies at different ciprofloxacin concentrations, by using anchored microfluidic drops and an image and data analysis pipelines. The microfluidic results are benchmarked against classical microbiology measurements of antibiotic susceptibility, showing an agreement between the pooled microfluidic chip and replated bulk measurements. Further, the experimental likelihood of a single cell to form a colony is used to provide a probabilistic antibiotic susceptibility curve. In addition to the probabilistic viewpoint, the microfluidic format enables the characterization of morphological features over time for a large number of individual cells. This pipeline can be used to compare the response of different bacterial strains to antibiotics with different action mechanisms.

While many cell culture systems are sensitive to the conditions in which cells are introduced into the system, we find that in situ differentiated tube-shaped microfluidic organoids have a particularly high sensitivity. Preliminary experiments using conventional seeding techniques revealed that biases in initial cell number and distribution dramatically impacted organoid shape and behavior downstream. Residual flows during seeding further complicated the process, dispersing cells to undesirable locations within the chip. To address this problem, a a robotic seeding system for controlling the process of inserting cells into microfluidic chips was developed. Environmental control of temperature, CO2, and humidity was implemented by modifying a commercial Arduino-controlled incubator. An eight-channel syringe pump controlled flow to eight cell dispensers, while a vertical leadscrew stage raised and lowered them, and a set of stackable flexure micromanipulators individually controlled the X and Y position of each cell dispenser. The flexure manipulators were 3D printed, driven by low-cost motors and electronics, and required little assembly and no alignment, resulting in a cheap and scalable method of controlling a dense array of micromanipulators. A dual objective microscope on a motorized gantry used an oblique lighting system to observe the seeding process, allowing for real-time interventions or passive observation of automated protocols. The robotic cell seeding system provided a platform for optimizing a sensitive process towards increasing the repeatability and physiological relevance of tube-shaped microfluidic organoids.

Transwell-based airway models have become increasingly important to study the effects of respiratory diseases and drug treatment at the air-liquid interface of the lung epithelial barrier. However, the underlying mechanisms at tissue and cell level often remain unclear, as transwell inserts feature limited live-cell imaging compatibility. Here, we report on a novel microphysiological platform for the cultivation of transwell-based lung tissues providing the possibility to alternate between air-liquid and liquid-liquid interfaces. While the air-liquid interface recapitulates physiological conditions for the lung model, the liquid-liquid interface enables live-imaging of the tissue at high spatiotemporal resolution. The plastics-based microfluidic platform enables insertion and recuperation of the transwell inserts, which allows for tissue cultivation and analysis under standardized well plate conditions. We used the device to monitor infections of Pseudomonas aeruginosa in human stem-cell-derived bronchial epithelial tissue. We continuously imaged the progression of a P. aeruginosa infection in real time at high resolution, which provided insights into bacterial spreading and invasion on the apical tissue surface, as well as insights into tissue breaching and destruction over time. The airway tissue culture system is a powerful tool to visualize and elucidate key processes of developing respiratory diseases and to facilitate drug testing and development.

The tumor microenvironment (TME) plays critical roles in cancer development, aggressiveness, and treatment resistance. The TME comprises cellular (stromal) components as well as gradients of physicochemical properties, including hypoxia and acidosis. Understanding of how hypoxia and acidosis gradients impact cancer phenotypes is lacking, in large part due to challenges in precisely mimicking and controlling such gradients in a manner compatible with the growth of cancer- and stromal cells. Here, we design and validate a microfluidic device enabling orthogonal gradients of oxygen and pH. Both gradients are established by diffusion from a nearby source and sink in the observation area in the absence of flow. This produces linear gradients at steady state. Our device enables a wide range of spatiotemporally resolved analyses, from omics to live cell imaging, interrogating the impact of the physicochemical TME on disease development. The design is easily adaptable, making it valuable for a wide range of questions involving physicochemical gradients.

High-throughput microfluidics has transformed biomedical research by enabling precise and parallel sample handling, but most devices are single-use due to channel occlusion and contamination from experiments. Alongside low fabrication yield and reduced experimental success associated with dense microfeatures, this creates a major bottleneck for scalable high-throughput applications. We present a rapid, reusable, and modular high-throughput microfluidic platform with integrated microvalves for automation. The platform employs a multilayer architecture consisting of a custom casing, PDMS layers with dense microfeatures for fluid handling and culture, and a glass substrate. Permanent bonding is applied only between control and fluid layers, while reversible bonding is used at all other interfaces, including the substrate. Because substrate is the primary cell-contact surface and can be readily detached, the remaining layers can be disassembled, thoroughly cleaned, and reused with minimal processing on a new substrate. This approach improves repeatability and experimental success while reducing preparation time from days to [~]2 hours. The disassemblable design also supports incorporation of application-specific layers between fluid layer and substrate, enhancing platform versatility for 3D culture. We validated performance through pressure/flow characterization and on-chip cell/organoid culture. Overall, our platform accelerates rapid high-throughput data generation across diverse biological applications.

Anti-cancer drugs have the lowest success rate of approval in drug development programs. Thus, preclinical assays that closely predict the clinical responses to drugs are of utmost importance in both clinical oncology and pharmaceutical research. 3D tumour models preserve the tumoural architecture and are cost-, labour-, and time-efficient. However, the short-term longevity, limited throughput, and limitations to live imaging of these models have so far driven researchers towards simpler, less realistic tumour models such as monolayer cell cultures. Here, we present a static open-space microfluidic drug screening platform that enables the formation, culture, and multiplexed delivery of several reagents to various 3D tumour models, namely cancer cell line spheroids and ex vivo primary tumour fragments. Our platform utilizes an open-space microfluidic technology, a pixelated chemical display, which creates fluidic "pixels" of biochemical reagents that stream over tumour models in a contact-free fashion. Up to 9 different treatment conditions can be tested over 144 samples in a single experiment. We provide a proof-of-concept application by staining fixed and live tumour models with multiple cellular dyes. Furthermore, we demonstrate that the various responses of the tumour models to biological stimuli can be assessed using the proposed drug screening platform. The platform is amenable to various 3D tumour models, such as tumour organoids. Upscaling of the microfluidic platform to larger areas can lead to higher throughputs, and thus will have a significant impact on developing treatments for cancer.

Hematogenous metastasis is initiated when tumor cells (TCs) intravasate into the vasculature, yet intravasation remains poorly understood because it is difficult to observe in vivo and intravasated TCs are challenging to isolate. To address these challenges, we developed IntravChip, a continuously perfused microfluidic platform containing a vascularized primary tumor microenvironment (TME) enabling the observation of TC intravasation, and a downstream chamber to collect intravasated TCs. The IntravChip can support a high TC concentration in the TME while maintaining complete vascular perfusion, which we found was necessary to collect intravasated cells. Using MDA-MB-231 breast TCs, we identified an optimal initial TC seeding density that, by day 9, yields a densely populated TME and 100-440 collected intravasated TCs. We validated the IntravChip across several TC types, showing that MDA-MB-231 and MV3 TCs have the highest intravasation rates while MCF-7 TCs have low intravasation efficiency. We also show that the IntravChip is compatible with super-resolution nano-imaging. Our devices enabled high-quality STORM imaging, which revealed that H3K9me3 nanodomains are significantly differentially distributed in intravasated MDA-MB-231 tumor cells compared to those residing in the TME. Finally, the IntravChip was validated as a platform to test the effects of anti-cancer drugs on tumor cells and on the vasculature. We showed that a 5 M concentration of sorafenib reduced intravasation events by 69% without impacting the morphology of the microvascular networks (MVNs), while a 10 M concentration led to a significant decrease in vessel diameter. This platform enables quantitative analysis of TC intravasation, collection of intravasated TCs for characterization, and screening of anti-metastatic therapies.

In vitro artificial colonic micro-devices that better replicate complex in vivo systems are essential tools for advancing our understanding of human gut (patho)physiology. Micro-physiological systems (MPS) offer controlled environments, enabling precise manipulation of tissue topography, rigidity and nutrient flow. We present the EnView system configured as a human colon-based MPS that faithfully replicates the 3D topography and matrix stiffness of the human colonic environment using an interpenetrating network of polyacrylamide and collagen I hydrogels, and enabling the culture of human colonic epithelium for weeks. The EnView system integrates a microfluidic chamber with active control of apical/luminal and basal/stromal compartments, allowing for in situ imaging and monitoring. Human colonic epithelial Caco-2 cells cultured up to 21 days in this system follow the crypt topography and formed a polarized epithelial monolayer. This innovative MPS recapitulates in vitro a human colon epithelium with its 3D matrix topology and stiffness control, integrates a microfluidic chamber allowing active control of the apical/luminal and basal/stromal compartments by accurate injection, as well as in situ imaging. TeaserHuman gut-on-chip reproducing tissue mechanical properties with active microfluidic control for 3D tissue characterization.

Immune-cancer cell interactions play a central role in understanding antitumor responses and evaluating immunotherapies. However, long-term, single-cell-level analysis of these interactions remains challenging. To address this, we developed a microfluidic trapping device with 1,024 traps, each equipped with a filter to retain cells, sustain medium flow, minimize cross-talk, and allow precise control of effector-to-target (E:T) ratios. The platform enables continuous monitoring of immune-cancer interactions for up to 14 hours. Device characterization was performed using 10 {micro}m fluorescent beads seeded via hydrostatic flow, with trap occupancy validated by Poisson statistics. Initial experiments using PBMCs against GFP-expressing U87 (U87GFP) glioblastoma cells demonstrated an immune-mediated reduction in GFP intensity, which was interpreted cautiously as a cytotoxic response. To improve reproducibility, we subsequently employed IL-2-stimulated Natural Killer cells (NK92IL2) as standardized effectors and evaluated their interactions with U87GFP glioblastoma cells, K562 chronic myelogenous leukemia cells, and LS174T adenocarcinoma cells. Time-lapse imaging revealed transient intracellular calcium fluxes, consistent with early activation of NK92IL2 cells, followed by a cytotoxic response. Increasing E:T ratios consistently enhanced immune activity, highlighting the utility of this device for dissecting immune-cancer interactions and guiding the development of immunotherapy.

The lower respiratory tract is a hierarchical network of compliant tubular structures that are made from extracellular matrix proteins with a wall lined by an epithelium. While microfluidic airway-on-a-chip models incorporate the effects of shear and stretch on the epithelium, week-long air-liquid-interface (ALI) culture remains limited to static conditions. The circular cross-section and substrate compliance associated with intact airways have yet to be recapitulated to allow studies of epithelial injuries under physiological and ventilation conditions. To overcome these limitations, we present a collagen tube-based airway model. Sustaining a functional human bronchial epithelium during two-week perfusion is accomplished by continuously supplying warm, humid air at the apical side and culture medium at the basal side. The model faithfully recapitulates human airways in size, composition, and mechanical microenvironment, allowing for the first time dynamic studies of elastocapillary phenomena associated with regular breathing as well as mechanical ventilation, along with the impact on epithelial cells. Findings reveal the epithelium to become increasingly damaged when subjected to repetitive collapse and reopening as opposed to overdistension and suggest expiratory flow resistance to reduce atelectasis. We expect the model to find broad potential applications in organ-on-a-chip applications for various tubular tissues.

Understanding how the brain processes sensory information and produces appropriate behavior is a fundamental question in neuroscience. In this study, we developed a novel microfluidic device that allows for simultaneous observation of neural activity and behavior in Caenorhabditis elegans (C. elegans) during chemosensory stimulation. Traditional methods often involve trade-offs between high-resolution neuronal imaging, behavioral recording, and the ability to apply chemical stimulation. Our innovative design overcomes these limitations by immobilizing the worms head, stabilizing neuronal imaging, while allowing the posterior portion of the body to move freely, enabling the study of naturalistic behaviors during chemical stimulation. We applied this device to investigate how C. elegans responds to both attractive and aversive chemical cues. By correlating neural activity with observed behavior, we identified neurons and whole-brain dynamics associated with specific movements. Our results demonstrate that providing the worm with greater freedom of movement results in more naturalistic neuronal and behavioral responses to stimuli, compared to fully immobilized setups. This new tool offers a powerful approach for studying how sensory information is processed in the C. elegans nervous system to generate behavior, with potential applications in other model organisms. Its versatility and ease of operation make this device broadly applicable for studying how neural circuits drive behavior and decision-making in complex environments.

Microfluidics devices are powerful tools for studying dynamic processes in live cells, especially when used in conjunction with light microscopy. There are many applications of microfluidics devices including recording dynamic cellular responses to small molecules or other chemical conditions in perfused media, monitoring cell migration in constrained spaces, or collecting media perfusate for the study of secreted compounds in response to experimental inputs/manipulations. Here we describe a configurable low-cost (channel-based) microfluidics platform for live-cell microscopy, intended to be useful for experiments that require more precision/flexibility than simple rubber spacers, but less precision than molded elastomer-based platforms. The materials are widely commercially available, low-cost, and device assembly takes only minutes.

Microwell microfluidics has emerged as powerful platforms for high-precision biological and chemical investigations, bridging microscale fluid handling with compartmentalized reaction environments. Achieving robust and reproducible performance in such studies requires substantial effort to optimize microwell array design. This burden could be markedly alleviated by the availability of a curated database of microwell array parameters. Such a resource would enable the application of machine-learning models for performance prediction and automated design, leveraging knowledge accumulated from prior microfluidics research. However, constructing such a database entails a considerable investment of time and extensive manual curation, as microwell performance is governed by numerous critical design parameters that are reported inconsistently across a broad and largely unstructured body of literature. In this study, we introduce MicrowellMicrofluidicsMiner (M3), a framework that employs large language model (LLM) agents for autonomous knowledge extraction in microwell microfluidics. To evaluate its performance, we curate a ground-truth database and establish an LLM-driven assessment approach. Our results demonstrate that M3 achieves a peak accuracy of approximately 78%, representing more than a twofold improvement over the lowest observed accuracy (32%) obtained using a standalone LLM model (LLAMA 3.1). This study provides a foundational reference for researchers seeking to apply LLM agents to data-driven microfluidics research. The insights presented have the potential to substantially improve how scientists across microfluidics-related disciplines access, interpret, and leverage scientific information, thereby accelerating the development of innovative microfluidic devices and associated discoveries.

Population heterogeneity in bacterial phenotypes, such as antibiotic resistance, is increasingly recognized as a medical concern. Heteroresistance (HR) occurs when a predominantly susceptible bacterial population harbors a rare resistant subpopulation. During antibiotic exposure, these resistant bacteria can be selected and lead to treatment failure. Standard antibiotic susceptibility testing (AST) methods often fail to reliably detect these subpopulations due to their low frequency, highlighting the need for new diagnostic approaches. Here, we present a droplet microfluidics method where bacteria are encapsulated in droplets containing growth medium and antibiotics. The growth of rare resistant cells is detected by observing droplet shrinkage under microscopy. We validated this method for three clinically important antibiotics in Escherichia coli isolates obtained from bloodstream infections and showed that it can detect resistant subpopulations as infrequent as 10-6 using only 200 to 300 droplets. Additionally, we designed a multiplex microfluidic chip to increase the throughput of the assay.

Organ-on-Chips (OoC) have the potential to revolutionize drug testing. However, the fragmented ecosystem of available OoC systems leads to wasted resources and collaboration barriers, slowing uptake. To address this, there is a need for OoC platforms based on interoperability standards, modularity, and reconfigurability. Technology platforms based on open designs would enable seamless integration of diverse OoC models and components, facilitating translation. Our study introduces a modular microfluidic platform that integrates swappable modules for pumping, sensing, and OoCs, all within the ANSI/SLAS microplate footprint. Sub-components operate as microfluidic building blocks (MFBBs) and can interface with the demonstrated Fluidic Circuit Board (FCB) universally as long as the designs adhere to ISO standards. The platform architecture allows tube-less inter-module interactions via arbitrary and reconfigurable fluidic circuits. We demonstrate two possible fluidic configurations which include in-line sensors and furthermore demonstrate biological functionality by running both in-vitro and ex-vivo OoC models for multiple days. This platform is designed to support automated multi-organ experiments, independent of OoC type or material. All designs shown are made open source to encourage broader compatibility and collaboration.

The pathology of human diseases are now investigated using microphysiological systems (MPS) supporting vascular structures. Efforts to increase physiological relevance of these platforms have centered on the incorporation of organ-specific cellular and non-cellular constituents. However, tissue-specific cellular constituents must experience appropriate physical forces to faithfully replicate physiological function. Quantification of physical forces in MPS has received little attention. The goal of this study was to establish a simple and robust system capable of interfacing with existing pumps to quantitatively characterize the flow delivered to a MPS. The system assessed both the fluid pressure driving flow through a microphysiological platform and the resistance to flow of glass capillary tubes or a model vascular network. The system showed excellent qualitative and quantitative agreement with resistance values measured by a hydrostatic approach and predicted for laminar flow through a smooth capillary tube. Importantly, the system is optically-based without sensors contacting the circulating fluid making it ideally-suited for long-term biological studies where sterility is paramount. Benchmarking experiments were supplemented with measurements of driving pressure and flow resistance from vascular structures within a MPS in a humidified incubator. Vascular resistance measurements were consistent with published results obtained from similar microvascular networks.

Microfluidics optimize experimental procedures but often require external pumps for precise, steady, and low flow rates. These procedures typically require extended, continuous operation for long-duration experiments. We introduce the Dual-Syringe Continuous Pumping Mechanism (DSCPM), a low-cost, precise, and continuous pump for microfluidic applications with input multiplexing capability. With a 3D-printed housing and standard components, the DSCPM is easy to fabricate and accessible. Operating at a microliter per minute flow rates, the DSCPM uses fluidic bridge rectification to combine syringe pump precision with continuous infusion. We validated laminar flow in microfluidic cell traps without disrupting microbial growth. COMSOL simulations confirmed safe shear stress levels. We also developed and tested fluidic multiplexers for greater modularity and automation. Addressing current pump limitations, such as discontinuity and high costs, the DSCPM can enhance experimental capabilities and promote efficiency and precision while increasing accessibility of hardware automation in many fields.

Conventional cuvette-based and microfluidics-based bacterial electroporation approaches have distinct advantages, but they are typically limited to relatively small sample volumes, reducing their utility for applications requiring high throughput such as the generation of mutant libraries. Here, we present a disposable, user-friendly microfluidic electroporation device capable of processing large volume bacterial samples yet requiring minimal device fabrication and straightforward operation. We demonstrate that the proposed device can outperform conventional cuvettes in a range of situations, including across Escherichia coli strains with a range of electroporation efficiencies, and we use its large-volume bacterial electroporation capability to generate a library of transposon mutants in the anaerobic gut commensal Bifidobacterium longum.

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.

Rapid and accurate sorting of biological samples is extremely useful in a wide variety of applications. The model nematode Caenorhabditis elegans lends itself to automated fluid-flow based sorting because of its ability to live in aqueous solutions. Here, we build upon previous developments to construct a microfluidic device capable of sorting individuals based on a variety of characteristics, with a specific application toward differentiating fluorescently marked individuals. We find that our new design generates highly repeatable pools of sorted individuals. In general, there tends to be a tradeoff between precision and speed that can be optimized based on several different factors. Importantly, sorting does not decrease offspring production, and individuals can be sorted multiple times for increased precision. We provide detailed parts lists, schematics and software to allow implementation of these methods in other laboratories. Our results demonstrate that custom-made sorter chips can be a flexible and versatile addition to the nematode experimental toolbox.