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This paper describes, in detail, a method that uses flow cytometry to quantitatively characterise the performance of continuous-flow microfluidic devices designed to separate particles. Whilst simple, this approach overcomes many of the issues with the current commonly utilised methods (high-speed fluorescent imaging, or cell counting via either a hemocytometer or a cell counter), as it can accurately assess device performance even in complex, high concentration mixtures in a way that was previously not possible. Uniquely, this approach takes advantage of pulse processing in flow cytometry to allow quantitation of cell separation efficiencies and resulting sample purities on both single cells as well as cell clusters (such as circulating tumour cell (CTC) clusters). Furthermore, it can readily be combined with cell surface phenotyping to measure separation efficiencies and purities in complex cell mixtures. This method will facilitate the rapid development of a raft of continuous flow microfluidic devices, will be helpful in testing novel separation devices for biologically relevant clusters of cells such as CTC clusters, and will provide a quantitative assessment of device performance in complex samples, which was previously impossible. [ABSTRACT FROM AUTHOR]

Background: Local recurrence and metastasis remain the major causes of death in head and neck cancer (HNC) patients. Circulating tumour cells (CTCs) are shed from primary and metastatic sites into the circulation system and have been reported to play critical roles in the metastasis and recurrence of HNC. Here, we explored the use of CTCs to predict the response to treatment and disease progression in HNC patients. Methods: Blood samples were collected at diagnosis from HNC patients (n = 119). CTCs were isolated using a spiral microfluidic device and were identified using immunofluorescence staining. Correlation of baseline CTC numbers to 13-week PET-CT data and multidisciplinary team consensus data were conducted. Results: CTCs were detected in 60/119 (50.4%) of treatment naïve HNC patients at diagnosis. Baseline CTC numbers were higher in stage III vs. stage I-II p16-positive oropharyngeal cancers (OPCs) and other HNCs (p = 0.0143 and 0.032, respectively). In addition, we found that baseline CTC numbers may serve as independent predictors of treatment response, even after adjusting for other conventional prognostic factors. CTCs were detected in 10 out of 11 patients exhibiting incomplete treatment responses. Conclusions: We found that baseline CTC numbers are correlated with treatment response in patients with HNC. The expression level of cell-surface vimentin (CSV) on CTCs was significantly higher in patients with persistent or progressive disease, thus providing additional prognostic information for stratifying the risk at diagnosis in HNC patients. The ability to detect CTCs at diagnosis allows more accurate risk stratification, which in the future may be translated into better patient selection for treatment intensification and/or de-intensification strategies. [ABSTRACT FROM AUTHOR]

Elasto-inertial microfluidics has drawn significant attention in recent years due to its enhanced capabilities compared to pure inertial systems in control of small microparticles. Previous investigations have focused mainly on the applications of elasto-inertial sorting, rather than studying its fundamentals. This is because of the complexity of simulation and analysis, due to the presence of viscoelastic force. There have been some investigative efforts on the mechanisms of elasto-inertial focusing in straight channels; however, these studies were limited to simple rectangular channels and neglected the effects of geometry and flow rates on focusing positions. Herein, for the first time, we experimentally and numerically explore the effects of elasticity accompanying channel cross-sectional geometry and sample flow rates on the focusing phenomenon in elasto-inertial systems. The results reveal that increasing the aspect ratio weakens the elastic force more than inertial force, causing a transition from one focusing position to two. In addition, they show that increasing the angle of a channel corner causes the elastic force to push the particles more efficiently toward the center over a larger area of the channel cross section. Following on from this, we proposed a new complex straight channel which demonstrates a tighter focusing band compared to other channel geometries. Finally, we focused Saccharomyces cerevisiae cells (3–5 μm) in the complex channel to showcase its capability in focusing small-size particles. We believe that this research work improves the understanding of focusing mechanisms in viscoelastic solutions and provides useful insights into the design of elasto-inertial microfluidic devices. [ABSTRACT FROM AUTHOR]

Overcoming neurite inhibition is integral for restoring neuronal connectivity after CNS injury. Actin dynamics are critical for neurite growth cone formation and extension. The tropomyosin family of proteins is a regarded as master regulator of actin dynamics. This study investigates tropomyosin isoform 3.1 (Tpm3.1) as a potential candidate for overcoming an inhibitory substrate, as it is known to influence neurite branching and outgrowth. We designed a microfluidic device that enables neurons to be grown adjacent to an inhibitory substrate, Nogo-66. Results show that neurons, overexpressing hTpm3.1, have an increased propensity to overcome Nogo-66 inhibition. We propose Tpm3.1 as a potential target for promoting neurite growth in an inhibitory environment in the central nervous system. [ABSTRACT FROM AUTHOR]

In this study, we report the use of a high-throughput microfluidic spiral chip to screen out eggs from a mixed age nematode population, which can subsequently be cultured to a desired developmental stage. For the sorting of a mixture containing three different developmental stages, eggs, L1 and L4, we utilized a microfluidic spiral chip with a trapezoidal channel to obtain a sorting efficiency of above 97% and a sample purity (SP) of above 80% for eggs at different flow rates up to 10 mL min−1. The result demonstrated a cost-effective, simple, and highly efficient method for synchronizing C. elegans at a high throughput (∼4200 organisms per min at 6 mL min−1), while eliminating challenges such as clogging and non-reusability of membrane-based filtration. Due to its simplicity, our method can be easily adopted in the C. elegans research community. [ABSTRACT FROM AUTHOR]

Bovine serum albumin (BSA) is widely used as an additive in polymerase chain reaction (PCR)-based microfluidic devices to passivate reactors and alleviate nucleic-acid amplification. BSA is available commercially in two types: either acetylated or non-acetylated. A survey of literature indicates that both types of BSA are used in PCR-based microfluidic devices. Our study results reveal that the use of acetylated BSA in PCR micro-devices leads to differential inhibition of PCR, compared to non-acetylated BSA. This result is noticed for the first time, and the differential inhibition generally goes un-noticed, as compared to complete PCR inhibition. [ABSTRACT FROM AUTHOR]

Diagnosis of malaria at the early stage of infection is challenging due to the difficulty in detecting low abundance parasites from blood. Molecular methods such as real-time polymerase chain reaction (qPCR) can be especially useful for detecting low parasitemia levels due to their high sensitivity and their ability to recognize different malarial species and strains. Unfortunately, the accuracy of qPCR-based malaria detection can be compromised by many factors, including the limited specificity of primers, presence of PCR inhibitors in blood serum and DNA contamination from nucleated blood cells. Here, we use a label-free, shear-modulated inertial microfluidic system to enrich malaria parasites from blood so as to facilitate a more reliable and specific PCR-based malaria detection. This technique capitalizes on cell focusing behaviors in high aspect ratio microchannels coupled with pinched flow dynamics to isolate ring-stage malaria parasites from lysed blood containing white blood cells (WBCs). In this high aspect ratio (ratio of the channel height to the width) platform, the high shear rate along the channel width causes the dispersed WBCs at the inlet to migrate and align into two streams near the channel sidewalls while the malaria parasites remain unfocused. Sensitive detection of parasites at spiked densities ranging from 103 to 104Plasmodium falciparum parasites per mL (~2–10 per μL) has been demonstrated; they have also been quantified in whole blood using qPCR. This is approximately 100-fold more sensitive than the gold standard conventional microscopy analysis of thick blood smears. The simplicity of this device makes it ideal for integration with an automatic system for ultra-fast and accurate detection despite low levels of parasitemia. It can also help in malaria screening and elimination efforts. [ABSTRACT FROM AUTHOR]

The detection and characterization of rare circulating tumor cells (CTCs) from the blood of cancer patients can potentially provide critical insights into tumor biology and hold great promise for cancer management. The ability to collect a large number of viable CTCs for various downstream assays such as quantitative measurements of specific biomarkers or targeted somatic mutation analysis is increasingly important in medical oncology. Here, we present a simple yet reliable microfluidic device for the ultra-high-throughput, label-free, size-based isolation of CTCs from clinically relevant blood volumes. The fast processing time of the technique (7.5 mL blood in less than 10 min) and the ability to collect more CTCs from larger blood volumes lends itself to a broad range of potential genomic and transcriptomic applications. A critical advantage of this protocol is the ability to return all fractions of blood (i.e., plasma (centrifugation), CTCs and white blood cells (WBCs) (size-based sorting)) that can be utilized for diverse biomarker studies or time-sensitive molecular assays such as RT-PCR. The clinical use of this biochip was demonstrated by detecting CTCs from 100% (10/10) of blood samples collected from patients with advanced-stage metastatic breast and lung cancers. The CTC recovery rate ranged from 20 to 135 CTCs mL−1 and obtained under high purity (of 1 CTC out of every 30–100 WBCs which gives ∼4 log depletion of WBCs). They were identified with immunofluorescence assays (pan-cytokeratin+/CD45−) and molecular probes such as HER2/neu. [ABSTRACT FROM AUTHOR]

Cancer cells continuously secrete inflammatory biomolecules which play significant roles in disease progression and tumor metastasis toward secondary sites. Despite recent efforts to capture cancer cells' intercellular secretion heterogeneity using microfluidics, the challenges in operation of these systems as well as the complexity of designing a biosensing assay for long-term and real-time measurement of single cell secretions have become grand research barriers. Here, we present a new capillary-based microfluidic biosensing approach to easily and reliably capture ~500 single cells inside isolated dead-end nanoliter compartments using simple pipette injection, and quantify their individual secretion dynamics at the single cell resolution over a long period of culture (~16 h). We first present a detailed investigation of the fluid mechanics underlying the formation of nanoliter compartments in the microfluidic system. Based on the measurement of single cell capture efficiency, we employ a one-step FRET-based biosensor which monitors the single cancer cells' protease activity. The sensor reports the fluorescent signal as a product of amino acid chain cleavage and reduction in its quenching capability. Using the single cell protease secretion data, we identified modes of cell secretion dynamics in our cell sample. While most of the cells had low secretion levels, two other smaller and more aggressive secretion dynamics were cells with secretion modes that include sharp spikes or slow but progressive trend. The method presented here overcomes the difficulties associated with performing single cell secretion assays, enabling a feasible and reliable technique for high throughput measurement of metabolic activities in cancer cells. Image 1 • A novel microfluidic biosensor for real-time monitoring of single cell secretion dynamics has been introduced. • The microfluidic device is designed based on capillary-assisted formation of nanoliter compartments. • One-step FRET-based biosensor has been employed to monitor single cancer cells' protease activity. • Using the single cell protease secretion data, we identified three clusters in the breast cancer cell sample. [ABSTRACT FROM AUTHOR]

Stem cells are specialised cells characterised by their unique ability to both self-renew and transform into a wide array of specialised cell types. The widespread interest in stem cells for regenerative medicine and cultivated meat has led to a significant demand for these cells in both research and practical applications. Despite the growing need for stem cell manufacturing, the industry faces significant obstacles, including high costs for equipment and maintenance, complicated operation, and low product quality and yield. Microfluidic technology presents a promising solution to the abovementioned challenges. As an innovative approach for manipulating liquids and cells within microchannels, microfluidics offers a plethora of advantages at an industrial scale. These benefits encompass low setup costs, ease of operation and multiplexing, minimal energy consumption, and the added advantage of being labour-free. This review presents a thorough examination of the prominent microfluidic technologies employed in stem cell research and explores their promising applications in the burgeoning stem cell industry. It thoroughly examines how microfluidics can enhance cell harvesting from tissue samples, facilitate mixing and cryopreservation, streamline microcarrier production, and efficiently conduct cell separation, purification, washing, and final cell formulation post-culture. [ABSTRACT FROM AUTHOR]

This study is an attempt to make a step forward to implement the very immature concept of pumpless transportation of liquid into a real miniaturized device or lab-on-chip (LOC) on a plastic substrate. "Inert" plastic materials such as polypropylene (PP) are used in a variety of biomedical applications but their surface engineering is very challenging. Here, it was demonstrated that with a facile innovative wettability patterning route using fluorosilanized UV-independent TiO2 nanoparticle coating it is possible to create wedge-shaped open microfluidic tracks on inert solid surfaces for low-cost biomedical devices (lab-on-plastic). For the future miniaturization and integration of the tracks into a device, a variety of characterization techniques were used to not only systematically study the surface patterning chemistry and topography but also to have a clear knowledge of its biological interactions and performance. The effect of such surface architecture on the biological performance was studied in terms of static/dynamic protein (bovine serum albumin) adsorption, bacterial (Staphylococcus aureus and Staphylococcus epidermidis) adhesion, cell viability (using HeLa and MCF-7 cancer cell lines as well as noncancerous human fibroblast cells), and cell patterning (Murine embryonic fibroblasts). Strategies are discussed for incorporating such a confined track into a diagnostic device in which its sensing portion is based on protein, microorganism, or cells. Finally, for the proof-of-principle of biosensing application, the well-known high-affinity molecular couple of BSA-antiBSA as a biological model was employed. [ABSTRACT FROM AUTHOR]

Application of microfluidic systems for the study of cellular behaviors has been a flourishing area of research in the past decade. In the process of probing cell biomechanics the passage of a cell through a narrow microchannel or a small pore has attracted much attention during the recent years. And the study of cellular deformability and transportability using these systems with enhanced resolution and accuracy has opened a new paradigm for high-throughput characterization of both healthy and diseased cell populations. Here we use the level-set method to explore the relationship between the transit time and mechanical properties of normal white blood cells (WBCs) and breast cancer epithelial cells (MCF7) under different microenvironmental parameters (i.e., pressure difference, cell size, effective cell surface tension, constriction size and taper angle) in a 2-D computational domain by considering the cell as a viscous drop. The novel biomechanical relations are obtained for each cell type by the Response Surface Method (RSM), relating microenvironmental parameters to the dimensionless entry time of the normal and cancer cells. Our results revealed that MCF7 cells show a significantly different behavior (a bifurcating behavior when the pressure difference of inlet/outlet increases) in regards to the dimensionless entry time as a function of microchannel taper angle in comparison with the WBC. These results suggest that the microenvironmental parameters have a significant effect on the transportability of the cells and different cells have different behaviors in response to a specific microenvironmental parameter. Finally, it can be claimed that this method can be also utilized to distinguish between benign and cancerous cells or even to probe tumor heterogeneity toward high throughput cell cytometry. [ABSTRACT FROM AUTHOR]