Next generation microfluidics: fulfilling the promise of lab-on-a-chip technologies.

Microfluidic lab-on-a-chip technologies enable the analysis and manipulation of small fluid volumes and particles at small scales and the control of fluid flow and transport processes at the microscale, leading to the development of new methods to address a broad range of scientific and medical challenges. Microfluidic and lab-on-a-chip technologies have made a noteworthy impact in basic, preclinical, and clinical research, especially in hematology and vascular biology due to the inherent ability of microfluidics to mimic physiologic flow conditions in blood vessels and capillaries. With the potential to significantly impact translational research and clinical diagnostics, technical issues and incentive mismatches have stymied microfluidics from fulfilling this promise. We describe how accessibility, usability, and manufacturability of microfluidic technologies should be improved and how a shift in mindset and incentives within the field is also needed to address these issues. In this report, we discuss the state of the microfluidic field regarding current limitations and propose future directions and new approaches for the field to advance microfluidic technologies closer to translation and clinical use. While our report focuses on using blood as the prototypical biofluid sample, the proposed ideas and research directions can be extrapolated to other areas of hematology, oncology, biology, and medicine.

Screen-Printed Sensors Modified with Nafion and Mesoporous Carbon for Electrochemical Detection of Lead in Blood.

Lead (Pb) has long been acknowledged as a systemic toxicant, with pronounced health impacts observed even at low exposure levels, particularly in children. Adverse effects include diminished cognitive function, altered behavior, and developmental delays. Consequently, it is imperative to conduct regular monitoring of Blood Lead Levels (BLLs). In this work, we report on an electrochemical sensor based on screen-printed carbon electrode (SPCE) coated with Nafion and mesoporous carbon (MC). The sensor system uses simple sample preparation (acidification and dilution of whole blood), minimal sample volume (a few blood drops, 200 µl), and swift time-to-results (1 h). A limit of quantitation (LOQ) of 0.3 µg dL-1 Pb was achieved in whole blood. To demonstrate the practical utility of our sensor system, we evaluated its performance in the analysis of blood samples collected from children (n = 25). Comparative analysis with the laboratory-based gold standard method of inductively coupled plasma mass spectrometry (ICP-MS) demonstrated approximately 77% accuracy and 94% precision. We anticipate that our approach will serve as a valuable tool for more frequent BLL monitoring, particularly in communities where access to laboratory testing is impractical or expensive.

Deciphering fibroblast-induced drug resistance in non-small cell lung carcinoma through patient-derived organoids in agarose microwells.

Patient-derived organoids (PDOs) serve as invaluable 3D tumor models, retaining the histological complexity and genetic heterogeneity found in primary tumors. However, the limitation of small sample volumes and the lack of tailored platforms have hindered the research using PDOs. Within the tumor microenvironment, cancer-associated fibroblasts play a pivotal role in influencing drug sensitivity. In this study, we introduce an agarose microwell platform designed for PDO-based tumor and tumor microenvironment models, enabling rapid drug screening and resistance studies with small sample volumes. These microwells, constructed using 3D printing molds, feature a U-shaped bottom and 200 µm diameter. We successfully generated co-culture spheroids of non-small cell lung carcinoma (NSCLC) cells, including NCI-H358 or A549, and NSCLC PDOs F231 or F671 with fibroblast cell line, WI-38. Our results demonstrate the production of uniformly-sized spheroids (coefficient of variation <30%), high viability (>80% after 1 week), and fibroblast-induced drug resistance. The PDOs maintained their viability (>81% after 2 weeks) and continued to proliferate. Notably, when exposed to adagrasib, a KRASG12C inhibitor, we observed reduced cytotoxicity in KRASG12C-mutant spheroids when co-cultured with fibroblasts or their supernatant. The fibroblast supernatant sustained proliferative signals in tumor models. Taking into account the physical features, viability, and drug resistance acquired through supernatants from the fibroblasts, our platform emerges as a suitable platform for in vitro tumor modeling and the evaluation of drug efficacy using patient-derived tissues.

trans-Endothelial neutrophil migration activates bactericidal function via Piezo1 mechanosensing.

The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.

Manganese in residential drinking water from a community-initiated case study in Massachusetts.

BACKGROUND: Manganese (Mn) is a metal commonly found in drinking water, but the level that is safe for consumption is unknown. In the United States (U.S.), Mn is not regulated in drinking water and data on water Mn concentrations are temporally and spatially sparse. OBJECTIVE: Examine temporal and spatial variability of Mn concentrations in repeated tap water samples in a case study of Holliston, Massachusetts (MA), U.S., where drinking water is pumped from shallow aquifers that are vulnerable to Mn contamination. METHODS: We collected 79 residential tap water samples from 21 households between September 2018 and December 2019. Mn concentrations were measured using inductively coupled plasma mass spectrometry. We calculated descriptive statistics and percent of samples exceeding aesthetic (secondary maximum containment level; SMCL) and lifetime health advisory (LHA) guidelines of 50 µg/L and 300 µg/L, respectively. We compared these concentrations to concurrent and historic water Mn concentrations from publicly available data across MA. RESULTS: The median Mn concentration in Holliston residential tap water was 2.3 µg/L and levels were highly variable (range: 0.03-5,301.8 µg/L). Mn concentrations exceeded the SMCL and LHA in 14% and 12% of samples, respectively. Based on publicly available data across MA from 1994-2022, median Mn concentration was 17.0 µg/L (N = 37,210; range: 1-159,000 µg/L). On average 40% of samples each year exceeded the SMCL and 9% exceeded the LHA. Samples from publicly available data were not evenly distributed between MA towns or across sampling years. IMPACT STATEMENT: This study is one of the first to examine Mn concentrations in drinking water both spatially and temporally in the U.S. Findings suggest that concentrations of Mn in drinking water frequently exceed current guidelines and occur at concentrations shown to be associated with adverse health outcomes, especially for vulnerable and susceptible subpopulations like children. Future studies that comprehensively examine exposure to Mn in drinking water and its associations with children's health are needed to protect public health.

Numerical Modeling of Physical Cell Trapping in Microfluidic Chips.

Microfluidic methods have proven to be effective in separation and isolation of cells for a wide range of biomedical applications. Among these methods, physical trapping is a label-free isolation approach that relies on cell size as the selective phenotype to retain target cells on-chip for follow-up analysis and imaging. In silico models have been used to optimize the design of such hydrodynamic traps and to investigate cancer cell transmigration through narrow constrictions. While most studies focus on computational fluid dynamics (CFD) analysis of flow over cells and/or pillar traps, a quantitative analysis of mechanical interaction between cells and trapping units is missing. The existing literature centers on longitudinally extended geometries (e.g., micro-vessels) to understand the biological phenomenon rather than designing an effective cell trap. In this work, we aim to make an experimentally informed prediction of the critical pressure for a cell to pass through a trapping unit as a function of cell morphology and trapping unit geometry. Our findings show that a hyperelastic material model accurately captures the stress-related softening behavior observed in cancer cells passing through micro-constrictions. These findings are used to develop a model capable of predicting and extrapolating critical pressure values. The validity of the model is assessed with experimental data. Regression analysis is used to derive a mathematical framework for critical pressure. Coupled with CFD analysis, one can use this formulation to design efficient microfluidic devices for cell trapping and potentially perform downstream analysis of trapped cells.

Microfluidic isolation of breast cancer circulating tumor cells from microvolumes of mouse blood.

Liquid biopsy has shown significant research and clinical implications in cancer. Particularly, the isolation of circulating tumor cells (CTCs) in preclinical studies can provide crucial information about disease progression and therefore may guide treatment decisions. Microfluidic isolation systems have played a considerable role in CTC isolation for cancer studies, disease diagnosis, and prognosis. CTCs are often studied using preclinical animal models such as xenografts or syngeneic models. However, most isolation systems are tested on human cell lines and human blood, whereas less validation studies are done on preclinical samples such as CTCs from mouse models. Here, we demonstrate and evaluate a complete workflow of a sized-based inertial microfluidic device to isolate CTCs from blood using exclusively mouse blood and mouse cancer cell lines. We then incorporate the cytospin, a commonly used method for enumeration of small number of cells in a glass slide to quantify the total cell yield of our workflow.

Single Cell Analysis of Inertial Migration by Circulating Tumor Cells and Clusters.

Single-cell analysis provides a wealth of information regarding the molecular landscape of the tumor cells responding to extracellular stimulations, which has greatly advanced the research in cancer biology. In this work, we adapt such a concept for the analysis of inertial migration of cells and clusters, which is promising for cancer liquid biopsy, by isolation and detection of circulating tumor cells (CTCs) and CTC clusters. Using high-speed camera tracking live individual tumor cells and cell clusters, the behavior of inertial migration was profiled in unprecedented detail. We found that inertial migration is heterogeneous spatially, depending on the initial cross-sectional location. The lateral migration velocity peaks at about 25% of the channel width away from the sidewalls for both single cells and clusters. More importantly, while the doublets of the cell clusters migrate significantly faster than single cells (~two times faster), cell triplets unexpectedly have similar migration velocities to doublets, which seemingly disagrees with the size-dependent nature of inertial migration. Further analysis indicates that the cluster shape or format (for example, triplets can be in string format or triangle format) plays a significant role in the migration of more complex cell clusters. We found that the migration velocity of a string triplet is statistically comparable to that of a single cell while the triangle triplets can migrate slightly faster than doublets, suggesting that size-based sorting of cells and clusters can be challenging depending on the cluster format. Undoubtedly, these new findings need to be considered in the translation of inertial microfluidic technology for CTC cluster detection.

Evolution of focused streams for viscoelastic flow in spiral microchannels.

Particle migration dynamics in viscoelastic fluids in spiral channels have attracted interest in recent years due to potential applications in the 3D focusing and label-free sorting of particles and cells. Despite a number of recent studies, the underlying mechanism of Dean-coupled elasto-inertial migration in spiral microchannels is not fully understood. In this work, for the first time, we experimentally demonstrate the evolution of particle focusing behavior along a channel downstream length at a high blockage ratio. We found that flow rate, device curvature, and medium viscosity play important roles in particle lateral migration. Our results illustrate the full focusing pattern along the downstream channel length, with side-view imaging yielding observations on the vertical migration of focused streams. Ultimately, we anticipate that these results will offer a useful guide for elasto-inertial microfluidics device design to improve the efficiency of 3D focusing in cell sorting and cytometry applications.

Three-dimensional lab-on-a-foil device for dielectrophoretic separation of cancer cells.

A simple, low-cost, three-dimensional (3D) lab-on-a-foil microfluidic device for dielectrophoretic separation of circulating tumor cells (CTCs) is designed and constructed. Disposable thin films are cut by xurography and microelectrode array are made with rapid inkjet printing. The multilayer device design allows the studying of spatial movements of CTCs and red blood cells (RBCs) under dielectrophoresis (DEP). A numerical simulation was performed to find the optimum driving frequency of RBCs and the crossover frequency for CTCs. At the optimum frequency, RBCs were lifted 120 µm in z-axis direction by DEP force, and CTCs were not affected due to negligible DEP force. By utilizing the displacement difference, the separation of CTCs (modeled with A549 lung carcinoma cells) from RBCs in z-axis direction was achieved. With the nonuniform electric field at optimized driving frequency, the RBCs were trapped in the cavities above the microchannel, whereas the A549 cells were separated with a high capture rate of 86.3% ± 0.2%. The device opens not only the possibility for 3D high-throughput cell separation but also for future developments in 3D cell manipulation through rapid and low-cost fabrication.

Elasto-Inertial Focusing Mechanisms of Particles in Shear-Thinning Viscoelastic Fluid in Rectangular Microchannels.

Growth of the microfluidics field has triggered numerous advances in focusing and separating microparticles, with such systems rapidly finding applications in biomedical, chemical, and environmental fields. The use of shear-thinning viscoelastic fluids in microfluidic channels is leading to evolution of elasto-inertial focusing. Herein, we showed that the interplay between the elastic and shear-gradient lift forces, as well as the secondary flow transversal drag force that is caused by the non-zero second normal stress difference, lead to different particle focusing patterns in the elasto-inertial regime. Experiments and 3D simulations were performed to study the effects of flowrate, particle size, and the shear-thinning extent of the fluid on the focusing patterns. The Giesekus constitutive equation was used in the simulations to capture the shear-thinning and viscoelastic behaviors of the solution used in the experiments. At low flowrate, with Weissenberg number Wi ~ O(1), both the elastic force and secondary flow effects push particles towards the channel center. However, at a high flowrate, Wi ~ O(10), the elastic force direction is reversed in the central regions. This remarkable behavior of the elastic force, combined with the enhanced shear-gradient lift at the high flowrate, pushes particles away from the channel center. Additionally, a precise prediction of the focusing position can only be made when the shear-thinning extent of the fluid is correctly estimated in the modeling. The shear-thinning also gives rise to the unique behavior of the inertial forces near the channel walls which is linked with the 'warped' velocity profile in such fluids.

Automatic Microtitrator for Small Volume Samples.

Electroanalytical sensors for point-of-care biomedical or point-of-use environmental sample analysis are gaining popularity due to low limits of detection, ease of miniaturization, convenience, and ability to work with small sample volumes. Since pH must be tightly controlled for optimum electrochemical performance, adjustment of pH in these samples is often a necessity. Yet manual titration is time-consuming and can be especially challenging for small volumes. End point determination can also be difficult. Current commercial automatic pH titrators are generally designed for large volume (>1 mL) batch titrations, while the existing microvolume titrators are semiautomatic at best, still relying on multiple manual steps. To address the gap, we developed an automatic microtitration system suitable for small volume samples. The system was validated using digested whole blood microsamples, successfully demonstrating accurate and rapid pH adjustment for samples as small as 100 µL. The simple modular construction of the system makes it compatible with acid washing for trace metal detection and other cleaning or sample preparation steps. The electrochemical detection of manganese heavy metal in blood at the parts per billion level showed no detectable contamination induced by the system. Ultimately, our simple, accurate, user-friendly automatic microtitration system can be used in the pH adjustment of microvolume samples and can potentially be extended to other pH end point analysis.

Microfluidic techniques for isolation, formation, and characterization of circulating tumor cells and clusters.

Circulating tumor cell (CTC) clusters that are shed from the primary tumor into the bloodstream are associated with a poor prognosis, elevated metastatic potential, higher proliferation rate, and distinct molecular features compared to single CTCs. Studying CTC clusters may give us information on the differences in the genetic profiles, somatic mutations, and epigenetic changes in circulating cells compared to the primary tumor and metastatic sites. Microfluidic systems offer the means of studying CTC clusters through the ability to efficiently isolate these rare cells from the whole blood of patients in a liquid biopsy. Microfluidics can also be used to develop in vitro models of CTC clusters and make possible their characterization and analysis. Ultimately, microfluidic systems can offer the means to gather insight on the complexities of the metastatic process, the biology of cancer, and the potential for developing novel or personalized therapies. In this review, we aim to discuss the advantages and challenges of the existing microfluidic systems for working with CTC clusters. We hope that an improved understanding of the role microfluidics can play in isolation, formation, and characterization of CTC clusters, which can lead to increased sophistication of microfluidic platforms in cancer research.

Electrochemical Determination of Manganese in Whole Blood with Indium Tin Oxide Electrode.

In this work, we demonstrate accurate and precise measurement of manganese (Mn) concentration in human whole blood with indium tin oxide (ITO) electrode using square wave stripping voltammetry. While an essential trace metal for human health, elevated levels of Mn due to environmental or occupational exposure have been associated with severe neuromotor dysfunction characterized by parkinsonism and cognitive dysfunction making the monitoring of Mn in whole blood necessary. Pediatric populations are particularly susceptible to Mn given their developing brain and potential long-term impacts on neurodevelopment. The current gold standard for whole blood Mn measurements is by ICP-MS, which is costly and time consuming. The electrochemical detection with ITO working electrode in this work showed a limit of detection of 0.5 µg l-1 and a linear range of 5 to 500 µg l-1, which encompasses the physiological Mn levels in human whole blood (5-18 µg l-1). Our results of Mn measurement in whole blood show an average precision of 96.5% and an average accuracy of 90.3% compared to ICP-MS for both the normal range (5-18 µg l-1) and the elevated levels (>36 µg l-1) that require medical intervention. These results demonstrate the feasibility of Mn measurements in human blood with electrochemical sensors.

Non-small cell lung carcinoma spheroid models in agarose microwells for drug response studies.

There is a growing interest in developing personalized treatment strategies for each cancer patient, especially those with non-small cell lung carcinoma (NSCLC) which annually accounts for the majority of cancer related deaths in the US. Yet identifying the optimal NSCLC treatment strategy for each cancer patient is critical due to a multitude of mutations, some of which develop following initial therapy and can result in drug resistance. A key difficulty in developing personalized therapies in NSCLC is the lack of clinically relevant assay systems that are suitable to evaluate drug sensitivity using a minuscule amount of patient-derived material available following biopsies. Herein we leverage 3D printing to demonstrate a platform based on miniature microwells in agarose to culture cancer cell spheroids. The agarose wells were shaped by 3D printing molds with 1000 microwells with a U-shaped bottom. Three NSCLC cell lines (HCC4006, H1975 and A549) were used to demonstrate size uniformity, spheroid viability, biomarker expressions and drug response in 3D agarose microwells. Results show that our approach yielded spheroids of uniform size (coefficient of variation <22%) and high viability (>83% after 1 week-culture). Studies using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKIs) drugs gefitinib and osimertinib showed clinically relevant responses. Based on the physical features, cell phenotypes, and responses to therapy of our spheroid models, we conclude that our platform is suitable for in vitro culture and drug evaluation, especially in cases when tumor sample is limited.

Acoustic bubble for spheroid trapping, rotation, and culture: a tumor-on-a-chip platform (ABSTRACT platform).

Cancer is the leading cause of death globally, with 90% of deaths being caused by cancer metastasis. Circulating tumor cells (CTCs) play an important role in early diagnosis of cancer metastasis and in monitoring of therapeutic response. Therefore, reliable methods to isolate, collect and culture CTCs are required to obtain information on metastasis status and therapeutic treatment. In this work, we present a CTC-processing system: acoustic bubble for spheroid trapping, rotation, and culture: a tumor-on-a-chip platform (ABSTRACT). The platform consists of a main channel, several parallel sub-microchannels with microcavities and culture chambers. The microcavity is designed to trap a bubble with desired shape at the entrance of the sub-microchannel. Under the acoustic actuation, the trapped bubble oscillates and creates a secondary radiation force to trap and rotate CTCs at a desired location. By controlling the acoustic bubble, CTCs can be continuously trapped from the blood flow, rotated to form a spheroid, and released to the microchamber for culture. We systematically investigated the effects of device geometry, flow parameters, and input voltage on trapping of CTCs to optimize the performance. Additionally, the successful on-chip spheroid culture demonstrates the biocompatibility and the simplicity of this platform. Besides simplifying conventional complex CTC processing procedures, this ABSTRACT platform also shows great potential for downstream analysis of tumor cells, such as monitoring the progression of metastasis and personalized drug testing.

Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFß Signaling via Activation of Calcineurin.

Control of ionic gradients is critical to maintain cellular homeostasis in both physiological and pathological conditions, but the role of ion channels in cancer cells has not been studied thoroughly. In this work we demonstrated that activity of the Kv11.1 potassium channel plays a vital role in controlling the migration of colon cancer cells by reversing the epithelial-to-mesenchymal transition (EMT) into the mesenchymal-to-epithelial transition (MET). We discovered that pharmacological stimulation of the Kv11.1 channel with the activator molecule NS1643 produces a strong inhibition of colon cancer cell motility. In agreement with the reversal of EMT, NS1643 treatment leads to a depletion of mesenchymal markers such as SNAIL1, SLUG, TWIST, ZEB, N-cadherin, and c-Myc, while the epithelial marker E-cadherin was strongly upregulated. Investigating the mechanism linking Kv11.1 activity to reversal of EMT into MET revealed that stimulation of Kv11.1 produced a strong and fast inhibition of the TGFß signaling. Application of NS1643 resulted in de-phosphorylation of the TGFß downstream effectors R-SMADs by activation of the serine/threonine phosphatase PP2B (calcineurin). Consistent with the role of TGFß in controlling cancer stemness, NS1643 also produced a strong inhibition of NANOG, SOX2, and OCT4 while arresting the cell cycle in G0/G1. Our data demonstrate that activation of the Kv11.1 channel reprograms EMT into MET by inhibiting TGFß signaling, which results in inhibition of motility in colon cancer cells.

Polycarbonate Masters for Soft Lithography.

Fabrication of microfluidic devices by soft lithography is by far the most popular approach due to its simplicity and low cost. The approach relies on casting of elastomers, such as polydimethylsiloxane (PDMS), on masters fabricated from photoresists on silicon substrates. These masters, however, can be expensive, complicated to fabricate, and fragile. Here we describe an optimized replica molding approach to preserve the original masters by heat molding of polycarbonate (PC) sheets on PDMS molds. The process is faster and simpler than previously reported methods and does not result in a loss of resolution or aspect ratio for the features. The generated PC masters were used to successfully replicate a wide range of microfluidic devices, including rectangular channels with aspect ratios from 0.025 to 7.3, large area spiral channels, and micropost arrays with 5 µm spacing. Moreover, fabrication of rounded features, such as semi-spherical microwells, was possible and easy. Quantitative analysis of the replicated features showed variability of <2%. The approach is low cost, does not require cleanroom setting or hazardous chemicals, and is rapid and simple. The fabricated masters are rigid and survive numerous replication cycles. Moreover, damaged or missing masters can be easily replaced by reproduction from previously cast PDMS replicas. All of these advantages make the PC masters highly desirable for long-term preservation of soft lithography masters for microfluidic devices.

Validation of Electrochemical Sensor for Determination of Manganese in Drinking Water.

Manganese (Mn) is an essential nutrient for metabolic functions, yet excessive exposure can lead to neurological disease in adults and neurodevelopmental deficits in children. Drinking water represents one of the routes of excessive Mn exposure. Both natural enrichment from rocks and soil, and man-made contamination can pollute groundwater that supplies drinking water for a substantial fraction of the U.S. population. Conventional methods for Mn monitoring in drinking water are costly and involve a long turn-around time. Recent advancements in electrochemical sensing, however, have led to the development of miniature sensors for Mn determination. These sensors rely on a cathodic stripping voltammetry electroanalytical technique on a miniaturized platinum working electrode. In this study, we validate these electrochemical sensors for the determination of Mn concentrations in drinking water against the standard method using inductively coupled plasma mass spectrometry (ICP-MS). Drinking water samples (n = 78) in the 0.03 ppb to 5.3 ppm range were analyzed. Comparisons with ICP-MS yielded 100% agreement, ∼70% accuracy, and ∼91% precision. We envision the use of our system for rapid and inexpensive point-of-use identification of Mn levels in drinking water, which is especially valuable for frequent monitoring where contamination is present.

Resolving dynamics of inertial migration in straight and curved microchannels by direct cross-sectional imaging.

The explosive development of inertial microfluidic systems for label-free sorting and isolation of cells demands improved understanding of the underlying physics that dictate the intriguing phenomenon of size-dependent migration in microchannels. Despite recent advances in the physics underlying inertial migration, migration dynamics in 3D is not fully understood. These investigations are hampered by the lack of easy access to the channel cross section. In this work, we report on a simple method of direct imaging of the channel cross section that is orthogonal to the flow direction using a common inverted microscope, providing vital information on the 3D cross-sectional migration dynamics. We use this approach to revisit particle migration in both straight and curved microchannels. In the rectangular channel, the high-resolution cross-sectional images unambiguously confirm the two-stage migration model proposed earlier. In the curved channel, we found two vertical equilibrium positions and elucidate the size-dependent vertical and horizontal migration dynamics. Based on these results, we propose a critical ratio of blockage ratio (ß) to Dean number (De) where no net lateral migration occurs (ß/De ∼ 0.01). This dimensionless number (ß/De) predicts the direction of lateral migration (inward or outward) in curved and spiral channels, and thus serves as a guideline in design of such channels for particle and cell separation applications. Ultimately, the new approach to direct imaging of the channel cross section enables a wealth of previously unavailable information on the dynamics of inertial migration, which serves to improve our understanding of the underlying physics.