Multi-channel Microfluidic Device Research Articles

Flow distribution in microchannel devices with U-shaped manifolds

This article investigates fluid distribution in multichannel microfluidic devices with U-shaped manifolds. The parameters investigated include Reynolds number, microchannel width, microchannel spacing width, and microchannel length. The study is carried out using a microfluidic device having 50 microchannels employing water as the working fluid. The flow distribution in the microfluidic device is investigated for microchannel widths between 100 µm to 500 µm and for Reynolds number ranging between 0.04 to 100. The effect of channel spacing width on flow distribution is investigated for microchannel spacing widths between 100 µm to 500 µm. The increase in Reynolds number affects the flow distribution by increasing the non-uniformity of the flow. It is found that increasing the microchannel width causes more non-uniformity in the flow. It is also found that increasing the microchannel spacing width decreased the non-uniformity of the flow distribution. It is found that increasing the channel width from 100 µm to 300 µm increased the flow maldistribution by 83%, and increasing the microchannel spacing width from 100 µm to 300 µm decreased the flow maldistribution by 42%. It is observed that the microchannel width has a stronger influence on the flow distribution compared with microchannel spacing width. The study is useful for examining the flow maldistribution in microfluidic devices as their performance is dependent on flow rate.

T Cells Chemotaxis Migration Studies with a Multi-Channel Microfluidic Device.

Immune surveillance is dependent on lymphocyte migration and targeted recruitment. This can involve different modes of cell motility ranging from random walk to highly directional environment-guided migration driven by chemotaxis. This study protocol describes a flow-based microfluidic device to perform quantitative multiplex cell migration assays with the potential to investigate in real time the migratory response of T cells at the population or single-cell level. The device also allows for subsequent in situ fixation and direct fluorescence analysis of the cells in the microchannel.

Centrifugal microfluidic platform with digital image analysis for parallel red cell antigen typing

This study presents a portable multichannel microfluidic device for parallel and digital analysis of red cell antigen typing. A zigzag-shaped precise metering channel was designed for the simultaneous aliquoting of samples, which is independent of the volume of the predeposited blood-typing reagents in the reaction chambers. The entire assay protocol can be conducted using a sequential-step spinning protocol, which resembles that of conventional tube tests for blood typing; however, the manual procedure is largely reduced compared to that of conventional systems. After loading the samples, the disc is centrifuged in a defined program with five sequential steps, each of which can be completed in a few seconds. Through step-wise centrifugation, predeposited antibodies react with red blood cells, enabling the parallel identification of multiple red blood cell antigens without cross-contamination in 1 min. This is combined with gentle mixing to rapidly concentrate the agglutinates, making both visual and digital determination of agglutination straightforward. A customized image analysis algorithm for automatically determining the agglutination state was developed to complement this microfluidic system. The acquired image is processed after the test. The blood type is determined using a machine learning algorithm based on a histogram of oriented gradients (HOG) and support vector machines (SVM). This allows digital analysis to mirror the classical laboratory procedure for blood-type determination more accurately. The system was trained using a validated dataset of 150 blood samples, presenting 750 different agglutination patterns. The combination of SVM and HOG achieved 94.10% in the micro-weighted performance evaluation. This integrated microfluidic chip-based platform provides a “sample-in and answer out” demonstration for red blood cell typing, ensuring fast and reliable results because minimum manual steps are involved.

Construction of 3D printed constructs based on microfluidic microgel for bone regeneration

For the fabrication of engineered tissue constructs by three-dimensional (3D) bioprinting technology, the cell viability will be significantly affected by the shear stress during extrusion process and the exposure of light for cross-linking. Microgels with independently controlled compartments were demonstrated to provide protection for cells encapsulation. Here, we proposed to prepare the core-shell structured microgels for cells encapsulation to prevent the cell damage from the shear stress in extrusion-based 3D printing processes. The core-shell structured microgels with core layer of type I collagen and shell layer of alginate were prepared using a one-step innovational microfluidics technology through a multichannel microfluidic device. In the microfluidic parameters, acetic acid concentration and flow rate ratios of water phase to oil phase were found to evidently affect the morphology of microgels and viability of encapsulated cells. Methacrylated silk fibroin (SilMA) and methacrylated gelatin (GelMA) were synthesized and blended with cell-laden microgels as bioinks to fabricate the 3D-printed constructs. The increasing content of SilMA would decrease the pore size and increase the compression property of SilMA/GelMA hydrogels. Importantly, the microgels-containing SilMA/GelMA construct ensure the improved cell proliferation as compared to the SilMA/GelMA counterpart. Furthermore, the in vivo experiments demonstrated that Microgels-15%SilMA/GelMA construct exhibited good biocompatibility and better bone formation performance compared with 15%SilMA/GelMA construct. Therefore, the strategy of preparing cell-laden microgels based on microfluidic technology to improve the survival rate of cells in the bioprinting process can be available and effective in development of tissue engineered constructs.

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear.

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. TheCandida clade, including Cp, is increasingly developing resistance to the first and the second line of antifungals. Cp is frequently isolated from hands and skin surfaces, as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to the body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and allow for the fluorescent quantification of individual isolates. We also demonstrate that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.

High blood flow shear stress values are associated with circulating tumor cells cluster disaggregation in a multi-channel microfluidic device.

Metastasis represents a dynamic succession of events involving tumor cells which disseminate through the organism via the bloodstream. Circulating tumor cells (CTCs) can flow the bloodstream as single cells or as multicellular aggregates (clusters), which present a different potential to metastasize. The effects of the bloodstream-related physical constraints, such as hemodynamic wall shear stress (WSS), on CTC clusters are still unclear. Therefore, we developed, upon theoretical and CFD modeling, a new multichannel microfluidic device able to simultaneously reproduce different WSS characterizing the human circulatory system, where to analyze the correlation between SS and CTC clusters behavior. Three physiological WSS levels (i.e. 2, 5, 20 dyn/cm2) were generated, reproducing values typical of capillaries, veins and arteries. As first validation, triple-negative breast cancer cells (MDA-MB-231) were injected as single CTCs showing that higher values of WSS are correlated with a decreased viability. Next, the SS-mediated disaggregation of CTC clusters was computationally investigated in a vessels-mimicking domain. Finally, CTC clusters were injected within the three different circuits and subjected to the three different WSS, revealing that increasing WSS levels are associated with a raising clusters disaggregation after 6 hours of circulation. These results suggest that our device may represent a valid in vitro tool to carry out systematic studies on the biological significance of blood flow mechanical forces and eventually to promote new strategies for anticancer therapy.

Application of an open-chamber multi-channel microfluidic device to test chemotherapy drugs

The use of precision medicine for chemotherapy requires the individualization of the therapeutic regimen for each patient. This approach improves treatment efficacy and reduces the probability of administering ineffective drugs. To ensure accurate decision-making in a timely manner, anticancer drug efficacy tests must be performed within a short timeframe using a small number of cancer cells. These requirements can be satisfied via microfluidics-based drug screening platforms, which are composed of complex fluidic channels and closed systems. Owing to their complexity, skilled manipulation is required. In this study, we developed a microfluidic platform, to accurately perform multiple drug efficacy tests using a small number of cells, which can be conducted via simple manipulation. As it is a small, open-chamber system, a minimal number of cells could be loaded through simple pipetting. Furthermore, the extracellular matrix gel inside the chamber provides an in vivo-like environment that enables the localized delivery of the drugs to spontaneously diffuse from the channels underneath the chamber without a pump, thereby efficiently and robustly testing the efficacy and resistance of multiple drugs. We demonstrated that this platform enabled the rapid and facile testing of multiple drugs using a small number of cells (~ 10,000) over a short period of time (~ 2 days). These results provide the possibility of using this powerful platform for selecting therapeutic medication, developing new drugs, and delivering personalized medicine to patients.

Prototyping a Versatile Two-Layer Multi-Channel Microfluidic Device for Direct-Contact Cell-Vessel Co-Culture.

Microfluidic devices are gaining increasing popularity due to their wide applications in various research areas. Herein, we propose a two-layer multi-channel microfluidic device allowing for direct-contact cell-vessel co-culture. Using the device, we built a co-culture model of the outer blood-retina barrier (oBRB), mimicking the in vivo retinal pigment epithelial cells-Bruch membrane-fenestrated choroids. To demonstrate the versatility of the design, we further modified the device by inserting platinum electrodes for trans-epithelial electrical resistance (TEER) measurement, demonstrating the feasibility of on-chip assessment of the epithelial barrier integrity. Our proposed design allows for direct-contact co-culture of cell–cell or cell–vessel, modifiable for real-time evaluation of the state of the epithelial monolayers.

Anthelmintic drug actions in resistant and susceptible C. elegans revealed by electrophysiological recordings in a multichannel microfluidic device

Many anthelmintic drugs used to treat parasitic nematode infections target proteins that regulate electrical activity of neurons and muscles: ion channels (ICs) and neurotransmitter receptors (NTRs). Perturbation of IC/NTR function disrupts worm behavior and can lead to paralysis, starvation, immune attack and expulsion. Limitations of current anthelmintics include a limited spectrum of activity across species and the threat of drug resistance, highlighting the need for new drugs for human and veterinary medicine. Although ICs/NTRs are valuable anthelmintic targets, electrophysiological recordings are not commonly included in drug development pipelines. We designed a medium-throughput platform for recording electropharyngeograms (EPGs)—the electrical signals emitted by muscles and neurons of the pharynx during pharyngeal pumping (feeding)—in Caenorhabditis elegans and parasitic nematodes. The current study in C. elegans expands previous work in several ways. Detecting anthelmintic bioactivity in drugs, compounds or natural products requires robust, sustained pharyngeal pumping under baseline conditions. We generated concentration-response curves for stimulating pumping by perfusing 8-channel microfluidic devices (chips) with the neuromodulator serotonin, or with E. coli bacteria (C. elegans’ food in the laboratory). Worm orientation in the chip (head-first vs. tail-first) affected the response to E. coli but not to serotonin. Using a panel of anthelmintics—ivermectin, levamisole and piperazine—targeting different ICs/NTRs, we determined the effects of concentration and treatment duration on EPG activity, and successfully distinguished control (N2) and drug-resistant worms (avr-14; avr-15; glc-1, unc-38 and unc-49). EPG recordings detected anthelmintic activity of drugs that target ICs/NTRs located in the pharynx as well as at extra-pharyngeal sites. A bus-8 mutant with enhanced permeability was more sensitive than controls to drug treatment. These results provide a useful framework for investigators who would like to more easily incorporate electrophysiology as a routine component of their anthelmintic research workflow.

E-cadherin mediated lateral interactions between neighbor cells necessary for collective migration

Collective cell movement is critical in pathological processes such as wound healing and cancer invasion. It entails complex interactions between adjacent cells and between cells-extracellular matrices. Most studies measure the migration patterns and force propagation by placing cells on flat, patterned substrates. The cooperative behavior resulting from cell-cell interactions is not well understood. We have developed a multi-channel microfluidic device that has junctional protein E-cadherin coated onto the sidewalls of the channels that enables the cells’ lateral interactions with their neighbors to be studied. Our study reveals that epithelial cells rely on lateral E-cadherin-based adhesions to maintain the cohesion of the group. Cells move faster in narrower channels, but the average velocity along the channels is reduced in E-cadherin coated channels versus non-adhesive channels. We have directly measured the forces in the cross-linking protein, alpha-actinin, using FRET sensors during cell migration, and found that higher tension exists at the cell edges adjacent to the walls coated with E-cadherin, the implication being E-cadherin transmits the shear forces but does not provide a driving force for this migration.

A novel microfluidic device to model the human proximal tubule and glomerulus

A novel multi-channel microfluidic device to model human glomerular filtration and proximal tubular re-uptake.

High-Throughput Contact Flow Lithography.

High-throughput fabrication of graphically encoded hydrogel microparticles is achieved by combining flow contact lithography in a multichannel microfluidic device and a high capacity 25 mm LED UV source. Production rates of chemically homogeneous particles are improved by two orders of magnitude. Additionally, the custom-built contact lithography instrument provides an affordable solution for patterning complex microstructures on surfaces.

Automated measurement of multiple cancer biomarkers using quantum-dot-based microfluidic immunohistochemistry.

We report an automated multiple biomarker measurement method for tissue from cancer patients using quantum dot (QD)-based protein detection combined with reference-based protein quantification and autofluorescence (AF) removal. For multiplexed detection of biomarkers in tissue samples, visualization of QDs on cytokeratin was performed to create a multichannel microfluidic device on sites with dense populations of tumor cells. Three major breast cancer biomarkers (i.e., estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) were labeled using QDs successively on cancer cells in tissue sections. For the automated measurement of biomarkers, a cytokeratin-based biomarker normalization method was used to measure the averaged expression of proteins. A novel AF-removal algorithm was developed, which normalizes the reference AF spectra reconstructed from unknown AF spectra based on random sampling. For accurate quantification of QDs, we automatically and accurately removed the AF signal from 344 spots of QD-labeled tissue samples using 240 reference AF spectra. Using analytical data with 10 tissue samples from breast cancer patients, the measured biomarker intensities were in good agreement with the results of conventional analyses.

A Localized Surface Plasmon Resonance Imaging Instrument for Multiplexed Biosensing

Localized surface plasmon resonance (LSPR) spectroscopy has been widely used for label-free, highly sensitive measurements of interactions at a surface. LSPR imaging (LSPRi) has the full advantages of LSPR but enables high-throughput, multiplexed measurements by simultaneously probing multiple individually addressable sensors on a single sample surface. Each spatially distinct sensor can be tailored to provide data regarding different surface functionalities or reaction environments. Previously, LSPRi has focused on single-particle sensing where the size scale is very small. Here, we create defined macroscale arrays of nanoparticles that are compatible with common patterning methods such as dip-pen nanolithography and multichannel microfluidic delivery devices. With this new LSPR sensing format, we report the first demonstration of multiplexed LSPR imaging and show that the increased throughput of our instrument enables the collection of a complete Langmuir binding curve on a single sensor surface. In addition, the multiplexed LSPR sensor is highly selective, as demonstrated by the hybridization of single-stranded DNA to complementary sequences immobilized on the sensor surface. The LSPR arrays described in this work exhibit uniform sensitivity and tailorable optical properties, making them an ideal platform for high-throughput, label-free analysis of a variety of molecular binding interactions.

Development of a microfluidic device for automated vitrification human embryo

Optimization cryopreservation protocols require loading/unloading cryoprotectants (CPAs) while minimizing negative osmotic effects on embryos, particularly in vitrification when high concentration of CPAs are employed. The current study applies microfluidic technology to minimize osmotic effects and improve overall outcome. Experimental lab study. A computer-controlled multi-channel microfluidic device and associated embryo trapping vitrification straw was developed to minimize osmotic effects. Donated frozen patient cleavage embryos were randomly divided into two groups. In conventional vitrification (n = 39) we used a stepwise method, the experimental group (n-34) used a computer controlled automatic microfluidic system which systematically mixing CPAs. Final vitrification CPAs loading solution is 30%CPAs (15% EG+15% DMSO+ 0.5M sucrose in 20% SSS (v/v in PBS) and initial unloading (thawing) from 1.0 M Sucrose to PBS with 20% SSS. Freezing and thawing solution samples were collected in 60 second intervals to examine osmotic change surrounding embryos for both groups. Day 4 and 5 development was assessed. Our data indicate that the automated vitrification system generated a more gradual osmolarity changes in comparison to the more abrupt osmolarity changes in the manual group during the both CPAs loading and unloading process. Post thawing, microfluidic vitrified/thawed embryos shows similar (P>0.05) embryo survival rates (85.3% vs 89.7%), morula formation rate (50.0% vs 46.2%), cavitation (41.2% vs 38.5%) and blastocyst formation rate (32.4% vs 30.8) compared to the control group. This computer-controlled automated vitrification system provides reduced osmotic effect evidenced by smoother osmotic changes for both CPAs loading /unloading procedures. This is the first automatic vitrification platform for with consistent and reproducible outcomes. It requires less, labor consuming and minimal inter-embryologist variability for human embryos cryopreservation.

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

A Polymer‐Based Nanopore‐Integrated Microfluidic Device for Generating Stable Bilayer Lipid Membranes

A parylene nanpore was built into multichannel microfluidic devices and a single protein channel was reconstituted in a bilayer lipid membrane (BLM) on the pore. Although BLMs on micropores used for channel recordings are usually too unstable for the exchange of solutions between the upper and lower sides, the BLMs formed in our nano­pores were stable enough to permit solution exchange.

Microfluidic Approach to Create Three-Dimensional Tissue Models for Biofilm-Related Infection of Orthopaedic Implants

With conventional in vitro culture methods, it is difficult to study complex interactions of host cells with pathogens and drugs in physiologically relevant microenvironments. To simulate orthopaedic implant-associated infection, a multi-channel microfluidic device was used to (1) observe in real-time the development of osteoblasts into three-dimensional (3D) tissue-like structures and (2) study how this development was influenced by phenotypes of Staphylococcus epidermidis. In the absence of bacteria, osteoblasts formed a confluent layer on the bottom channel surface, gradually migrated to the side and top surfaces, and formed calcified 3D nodular structures in 8 days. The delivery timing and concentration of an antibiotic were controlled to produce small colony variants, sessile biofilms, or dead cells of S. epidermidis. In the presence of the small colony variants, osteoblasts initially adhered, and spread, but were killed within 2 days. In contrast, the sessile biofilms and dead bacteria cells did not significantly interfere with the formation of tissue-like structures. The results suggest the possibility of creating in vitro tissue-biofilm-biomaterial interfaces and therefore 3D tissue models, as an entirely new method of studying biofilm-related infection of orthopaedic implants with physiological relevance.

Multi-channel microfluidic devices combined with electrospray ionization quadrupole time-of-flight mass spectrometry applied to the monitoring of glutamate release from neuronal cells

This paper describes an integrated system combining microfluidic devices with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) for monitoring cellular chemical release. To demonstrate the feasibility of this new system, the reported carnosine-protection process against Abeta42-induced glutamate released from PC12 cells, was monitored. Poly-L-lysine coated microchannels were used to culture cells. A multi-channel miniature extraction chip (MEC) was integrated into the design to remove salts and protein interference effects. ESI-Q-TOF-MS was employed to realize semi-quantitative and highly sensitive qualitative analysis. The protective effect of carnosine against Abeta42-induced neurotoxicity was evaluated under different conditions in microchannels in parallel. The secretion product analysis, carried out by ESI-Q-TOF-MS, was accomplished in 5 min using only 2.5 microL of solvent. Furthermore, we show that integrated microfluidic devices have significant potential for the analysis of cellular secretions, as well as for medical screening tests and for the diagnosis of specific diseases.