Review of cell and particle trapping in microfluidic systems

The ability to obtain ideal conditions for well-defined chemical microenvironments and controlled temporal chemical and/or thermal variations holds promise of high-resolution cell response studies, cell-cell interactions or e.g. proliferation conditions for stem cells. It is a major motivation for the rapid increase of lab-on-a-chip based cell biology research. In view of this, new chip-integrated technologies are at an increasing rate being presented to the research community as potential tools to offer spatial control and manipulation of cells in microfluidic systems. This is becoming a key area of interest in the emerging lab-on-a-chip based cell biology research field. This review focuses on the different technical approaches presented to enable trapping of particles and cells in microfluidic system.     

A microfluidic array with cellular valving for single cell co-culture

We present a highly parallel microfluidic approach for contacting single cell pairs. The approach combines a differential fluidic resistance trapping method with a novel cellular valving principle for homotypic and heterotypic single cell co-culturing. Differential fluidic resistance was used for sequential single cell arraying, with the adhesion and flattening of viable cells within the microstructured environment acting to produce valves in the open state. Reversal of the flow was used for the sequential single cell arraying of the second cell type. Plasma stencilling, along the linear path of least resistance, was required to confine the cells within the trap regions. Prime flow conditions with minimal shear stress were identified for highly efficient cell arraying (～99%) and long term cell culture. Larger trap dimensions enabled the highest levels of cell pairing (～70%). The single cell co-cultures were in close proximity for the formation of connexon structures and the study of contact modes of communication. The research further highlights the possibility of using the natural behaviour of cells as the working principle behind responsive microfluidic elements.     

A programmable microfluidic cell array for combinatorial drug screening

We describe the development of a fully automatic and programmable microfluidic cell culture array that integrates on-chip generation of drug concentrations and pair-wise combinations with parallel culture of cells for drug candidate screening applications. The device has 64 individually addressable cell culture chambers in which cells can be cultured and exposed either sequentially or simultaneously to 64 pair-wise concentration combinations of two drugs. For sequential exposure, a simple microfluidic diffusive mixer is used to generate different concentrations of drugs from two inputs. For generation of 64 pair-wise combinations from two drug inputs, a novel time dependent variable concentration scheme is used in conjunction with the simple diffusive mixer to generate the desired combinations without the need for complex multi-layer structures or continuous medium perfusion. The generation of drug combinations and exposure to specific cell culture chambers are controlled using a LabVIEW interface capable of automatically running a multi-day drug screening experiment. Our cell array does not require continuous perfusion for keeping cells exposed to concentration gradients, minimizing the amount of drug used per experiment, and cells cultured in the chamber are not exposed to significant shear stress continuously. The utility of this platform is demonstrated for inducing loss of viability of PC3 prostate cancer cells using combinations of either doxorubicin or mitoxantrone with TRAIL (TNF-alpha Related Apoptosis Inducing Ligand) either in a sequential or simultaneous format. Our results demonstrate that the device can capture the synergy between different sensitizer drugs and TRAIL and demonstrate the potential of the microfluidic cell array for screening and optimizing combinatorial drug treatments for cancer therapy.     

Gentle cell trapping and release on a microfluidic chip by in situ alginate hydrogel formation

Microfluidic devices are increasingly used to perform biological experiments on a single-cell basis. However, long-term stability of cell positions is still an issue. A novel biocompatible method for cell entrapment and release on a microchip is presented. It is based on the controlled formation of an alginate hydrogel by bringing two laminar flows of alginate and calcium ions in the range of 2 mM to 40 mM into contact. The resulting growth of a gel bar is used to enclose and immobilize yeast cells. Adding ethylenediaminetetraacetic acid (EDTA) to the alginate solution allows for control of the hydrogel growth, and by varying the ratio of Ca(2+) to EDTA concentrations gel growth or gel shrinkage can be induced at will. Trapped cells are released during shrinkage of the gel. The trapping efficiency for different cell speeds is investigated and the properties of gel growth are discussed using a diffusion model. Precise positioning of a single cell is demonstrated. The technique presented allows not only the reversible immobilization of cells under gentle conditions but also offers the potential of long-term cell cultures as shown by on-chip incubation of yeast cells. The procedure may provide a simple and fully biocompatible technique for a multitude of innovative experiments on cells in microsystems.     

A microfluidic microbial fuel cell array that supports long-term multiplexed analyses of electricigens

Microbial fuel cells (MFCs) are green energy technologies that exploit microbial metabolism to generate electricity. The widespread implementation of MFC technologies has been stymied by their high cost and limited power. MFC arrays in which device configurations or microbial consortia can be screened have generated significant interest because of their potential for defining aspects that will improve performance featuring high throughput characteristics. However, current miniature MFCs and MFC array systems do not support long-term studies that mimic field conditions, and hence, have limitations in fully characterizing and understanding MFC performances in varieties of conditions. Here, we describe an MFC array device that incorporates microfluidic technology to enable continuous long-term analysis of MFC performance at high throughput utilizing periodic anolyte/catholyte replenishment. The system showed 360% higher power output and 700% longer operating time when compared to MFC arrays without catholyte replenishment. We further demonstrate the utility of the system by reporting its successful use in screening microbial consortia collected from geographically diverse environments for communities that support enhanced MFC performance. Taken together, this work demonstrates that anolyte/catholyte replenishment can significantly improve the long-term performance of microfabricated MFC arrays, and support the characterization of diverse microbial consortia.     

Addressing a vascular endothelium array with blood components using underlying microfluidic channels

Here, we show that an array of endothelial cells, addressable by an underlying microfluidic network of channels containing red blood cells, can be employed as an in vitro model of in vivo circulation to monitor cellular communication between different cell types in the drug discovery process.     

Programmable large area digital microfluidic array with integrated droplet sensing for bioassays

We describe a new device concept for digital microfluidics, based on an active matrix electrowetting on dielectric (AM-EWOD) device. A conventional EWOD device is limited by the number of electrical connections that can be made practically, which restricts the number and type of droplet operations. In an AM-EWOD, the patterned electrodes of a conventional EWOD device are replaced by a thin film transistor (TFT) array, as found in a liquid crystal display (LCD), facilitating independent control of each electrode. The arrays can have many thousand individually addressable electrodes, are fully reconfigurable and can be programmed to support multiple simultaneous operations. Each element is 210 μm × 210 μm in size and contains a circuit that measures the electrical impedance of the liquid above it. This is used to determine the presence and size of a droplet, a method that can improve assay reliability and accuracy. This sensor provides feedback, error detection and closed loop control of an assay sequence. We describe the design, fabrication and testing of a 64 × 64 format AM-EWOD device with impedance sensor functionality. A colorimetric assay is implemented on the device and used to measure glucose in human blood serum. Results are compared with the same assay performed on a microtitre plate.     

A light writable microfluidic "flash memory": optically addressed actuator array with latched operation for microfluidic applications

This paper presents a novel optically addressed microactuator array (microfluidic "flash memory") with latched operation. Analogous to the address-data bus mediated memory address protocol in electronics, the microactuator array consists of individual phase-change based actuators addressed by localized heating through focused light patterns (address bus), which can be provided by a modified projector or high power laser pointer. A common pressure manifold (data bus) for the entire array is used to generate large deflections of the phase change actuators in the molten phase. The use of phase change material as the working media enables latched operation of the actuator array. After the initial light "writing" during which the phase is temporarily changed to molten, the actuated status is self-maintained by the solid phase of the actuator without power and pressure inputs. The microfluidic flash memory can be re-configured by a new light illumination pattern and common pressure signal. The proposed approach can achieve actuation of arbitrary units in a large-scale array without the need for complex external equipment such as solenoid valves and electrical modules, which leads to significantly simplified system implementation and compact system size. The proposed work therefore provides a flexible, energy-efficient, and low cost multiplexing solution for microfluidic applications based on physical displacements. As an example, the use of the latched microactuator array as "normally closed" or "normally open" microvalves is demonstrated. The phase-change wax is fully encapsulated and thus immune from contamination issues in fluidic environments.     

A microfluidic fuel cell with flow-through porous electrodes

A microfluidic fuel cell architecture incorporating flow-through porous electrodes is demonstrated. The design is based on cross-flow of aqueous vanadium redox species through the electrodes into an orthogonally arranged co-laminar exit channel, where the waste solutions provide ionic charge transfer in a membraneless configuration. This flow-through architecture enables improved utilization of the three-dimensional active area inside the porous electrodes and provides enhanced rates of convective/diffusive transport without increasing the parasitic loss required to drive the flow. Prototype fuel cells are fabricated by rapid prototyping with total material cost estimated at 2 USD/unit. Improved performance as compared to previous microfluidic fuel cells is demonstrated, including power densities at room temperature up to 131 mW cm-2. In addition, high overall energy conversion efficiency is obtained through a combination of relatively high levels of fuel utilization and cell voltage. When operated at 1 microL min-1 flow rate, the fuel cell produced 20 mW cm-2 at 0.8 V combined with an active fuel utilization of 94%. Finally, we demonstrate in situ fuel and oxidant regeneration by running the flow-through architecture fuel cell in reverse.     

An air-breathing microfluidic fuel cell with a finny anode

Using COMSOL multiphysics software and a previously validated 3D numerical model, performances of a novel air-breathing microfluidic fuel cell (MFFC) are discussed. The microfluidic fuel cell employs a simple structure composed of a flow channel with sloped upper wall, a gas diffusion cathode and a finny anode. Furthermore it can reduce the losses related to mixing in fuel-electrolyte interface and also promote the replenishment of the depletion layer on anode surfaces. Thus, high fuel utilization can be achieved. Numerical simulations show that the fuel utilization can be up to 70%.     

Enhancing cell recognition by scrutinizing cell surfaces with a nanoparticle array

We report a dual-ligand nanoparticle array approach for discerning cells that have different surface receptor profiles surrounding a common primary receptor expressed at high or low levels. The achieved differentiation provides nanoparticles the ability for potential applications in treatment of patients at a personalized medicine level for drug delivery and radiation therapy with a much better safety profile.     

Engineering of a microfluidic cell culture platform embedded with nanoscale features

Cells residing in a microenvironment interact with the extracellular matrix (ECM) and neighboring cells. The ECM built from biomacromolecules often includes nanotopography. Through the ECM, interstitial flows facilitate transport of nutrients and play an important role in tissue maintenance and pathobiology. To create a microenvironment that can incorporate both nanotopography and flow for studies of cell-matrix interactions, we fabricated microfluidic channels endowed with nanopatterns suitable for dynamic culture. Using polymer thin film technology, we developed a versatile stitching technique to generate a large area of nanopatterned surface and a simple microtransfer assembly technique to assemble polydimethylsiloxane-based microfluidics. The cellular study showed that both nanotopography and fluid shear stress played a significant role in adhesion, spreading, and migration of human mesenchymal stem cells. The orientation and deformation of cytoskeleton and nuclei were regulated through the interplay of these two cues. The nanostructured microfluidic platform provides a useful tool to promote the fundamental understanding of cell-matrix interactions and may be used to regulate the fate of stem cells.     

Analyzing cell mechanics in hematologic diseases with microfluidic biophysical flow cytometry

Pathological processes in hematologic diseases originate at the single-cell level, often making measurements on individual cells more clinically relevant than population averages from bulk analysis. For this reason, flow cytometry has been an effective tool for single-cell analysis of properties using light scattering and fluorescence labeling. However, conventional flow cytometry cannot measure cell mechanical properties, alterations of which contribute to the pathophysiology of hematologic diseases such as sepsis, diabetic retinopathy, and sickle cell anemia. Here we present a high-throughput microfluidics-based 'biophysical' flow cytometry technique that measures single-cell transit times of blood cell populations passing through in vitro capillary networks. To demonstrate clinical relevance, we use this technique to characterize biophysical changes in two model disease states in which mechanical properties of cells are thought to lead to microvascular obstruction: (i) sepsis, a process in which inflammatory mediators in the bloodstream activate neutrophils and (ii) leukostasis, an often fatal and poorly understood complication of acute leukemia. Using patient samples, we show that cell transit time through and occlusion of microfluidic channels is increased for both disease states compared to control samples, and we find that mechanical heterogeneity of blood cell populations is a better predictor of microvascular obstruction than average properties. Inflammatory mediators involved in sepsis were observed to significantly affect the shape and magnitude of the neutrophil transit time population distribution. Altered properties of leukemia cell subpopulations, rather than of the population as a whole, were found to correlate with symptoms of leukostasis in patients-a new result that may be useful for guiding leukemia therapy. By treating cells with drugs that affect the cytoskeleton, we also demonstrate that their transit times could be significantly reduced. Biophysical flow cytometry offers a low-cost and high-throughput diagnostic and drug discovery platform for hematologic diseases that affect microcirculatory flow.     

Electrode calibration with a microfluidic flow cell for fast-scan cyclic voltammetry

Fast-scan cyclic voltammetry (FSCV) is a common analytical electrochemistry tool used to measure chemical species. It has recently been adapted for measurement of neurotransmitters such as dopamine in awake and behaving animals (in vivo). Electrode calibration is an essential step in FSCV to relate observed current to concentration of a chemical species. However, existing methods require multiple components, which reduce the ease of calibrations. To this end, a microfluidic flow cell (μFC) was developed as a simple device to switch between buffer and buffer with a known concentration of the analyte of interest--in this case dopamine--in a microfluidic Y-channel. The ability to quickly switch solutions yielded electrode calibrations with faster rise times and that were more stable at peak current values. The μFC reduced the number of external electrical components and produced linear calibrations over a range of concentrations. To demonstrate this, an electrode calibrated with the μFC was used in FSCV recordings from a rat during the delivery of food reward--a stimulus that reliably evokes a brief increase in current due to the oxidation of dopamine. Using the linear calibration, dopamine concentrations were determined from the current responses evoked during the behavioral task. The μFC is able to easily and quickly calibrate FSCV electrode responses to chemical species for both in vitro and in vivo experiments.     

A novel high aspect ratio microfluidic design to provide a stable and uniform microenvironment for cell growth in a high throughput mammalian cell culture array

We present a high aspect ratio microfluidic device for culturing cells inside an array of microchambers with continuous perfusion of medium. The device was designed to provide a potential tool for cost-effective and automated cell culture. The single unit of the array consists of a circular microfluidic chamber 40 microm in height surrounded by multiple narrow perfusion channels 2 microm in height. The high aspect ratio (approximately 20) between the microchamber and the perfusion channels offers advantages such as localization of the cells inside the microchamber as well as creating a uniform microenvironment for cell growth. Finite element methods were used to simulate flow profile and mass transfer of the device. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C and was grown to confluency. The microfluidic cell culture array could potentially offer an affordable platform for a wide range of applications in high throughput cell-based screening, bioinformatics, synthetic biology, quantitative cell biology, and systems biology.     

Parallel separation of multiple samples with negative pressure sample injection on a 3-D microfluidic array chip

A simple and powerful microfluidic array chip-based electrophoresis system, which is composed of a 3-D microfluidic array chip, a microvacuum pump-based negative pressure sampling device, a high-voltage supply and an LIF detector, was developed. The 3-D microfluidic array chip was fabricated with three glass plates, in which a common sample waste bus (SW(bus)) was etched in the bottom layer plate to avoid intersecting with the separation channel array. The negative pressure sampling device consists of a microvacuum air pump, a buffer vessel, a 3-way electromagnet valve, and a vacuum gauge. In the sample loading step, all the six samples and buffer solutions were drawn from their reservoirs across the injection intersections through the SW(bus) toward the common sample waste reservoir (SW(T)) by negative pressure. Only 0.5 s was required to obtain six pinched sample plugs at the channel crossings. By switching the three-way electromagnetic valve to release the vacuum in the reservoir SW(T), six sample plugs were simultaneously injected into the separation channels by EOF and electrophoretic separation was activated. Parallel separations of different analytes are presented on the 3-D array chip by using the newly developed sampling device.     

Hydrodynamic simulation of cell docking in microfluidic channels with different dam structures

The immobilization of biological cells in micro-devices requires high efficiency and easy control while maintaining cell viability. One approach for cell immobilization is to utilize constriction structures such as dams to trap cells in microfluidics. In this paper, we present a comprehensive hydrodynamic analysis of two different types of constriction structures for cell immobilization: dams either in perpendicular or in parallel to the main flow route. Various structural models and experimental conditions were compared for cell docking and alignment, and the pressure and velocity profiles of the flow in the micro-channels and the hydrodynamic force and shear stress on the docked cells were calculated based on fluid dynamic theory and numerical simulation. The effects of the dam structures and cell docking on the flow properties, the transportation efficiency, and the induced stress on the docked cells were analyzed. Improved hydraulic pressure profiles in the auxiliary inlets were discussed for the modulation of the flow characteristics and attenuation of hydrodynamic forces exerted on the cells. Furthermore, a new design combining the advantages of perpendicular and parallel dam structures was proposed for cell-based microfluidics.     

A passive microfluidic hydrogen-air fuel cell with exceptional stability and high performance

We describe an advanced microfluidic hydrogen-air fuel cell (FC) that exhibits exceptional durability and high performance, most notably yielding stable output power (>100 days) without the use of an anode-cathode separator membrane. This FC embraces an entirely passive device architecture and, unlike conventional microfluidic designs that exploit laminar hydrodynamics, no external pumps are used to sustain or localize the reagent flow fields. The devices incorporate high surface area/porous metal and metal alloy electrodes that are embedded and fully immersed in liquid electrolyte confined in the channels of a poly(dimethylsiloxane) (PDMS)-based microfluidic network. The polymeric network also serves as a self-supporting membrane through which oxygen and hydrogen are supplied to the cathode and alloy anode, respectively, by permeation. The operational stability of the device and its performance is strongly dependent on the nature of the electrolyte used (5 M H2SO4 or 2.5 M NaOH) and composition of the anode material. The latter choice is optimized to decrease the sensitivity of the system to oxygen cross-over while still maintaining high activity towards the hydrogen oxidation reaction (HOR). Three types of high surface area anodes were tested in this work. These include: high-surface area electrodeposited Pt (Pt); high-surface area electrodeposited Pd (Pd); and thin palladium adlayers supported on a "porous" Pt electrode (Pd/Pt). The FCs display their best performance in 5 M H2SO4 using the Pd/Pt anode. This exceptional stability and performance was ascribed to several factors, namely: the high permeabilities of O2, H2, and CO2 in PDMS; the inhibition of the formation of insoluble carbonate species due to the presence of a highly acidic electrolyte; and the selectivity of the Pd/Pt anode toward the HOR. The stability of the device for long-term operation was modeled using a stack of three FCs as a power supply for a portable display that otherwise uses a 3 V battery.     

A radial microfluidic concentration gradient generator with high-density channels for cell apoptosis assay

An integrated microfluidic concentration gradient chip was developed for generating stepwise concentrations in high-density channels and applied to high-throughput apoptosis analysis of human uterine cervix cancer (HeLa) cells. The concentration gradient was generated by repeated splitting-and-mixing of the source solutions in a radial channel network which consists of multiple concentric circular channels and an increasing number of branch channels. The gradients were formed over hundreds of branches with predictable concentrations in each branch channel. This configuration brings about some distinctive advantages, e.g., more compact and versatile design, high-density of channels and wide concentration ranges. This concentration gradient generator was used in perfusion culture of HeLa cells and a drug-induced apoptosis assay, demonstrated by investigating the single and combined effects of two model anticancer drugs, 5-fluorouracil and Cyclophosphamide, which were divided into 65 concentrations of the two drugs respectively and 65 of their combinatorial concentrations. The gradient generation, the cell culture/stimulation and staining were performed in a single chip. The present device offers a unique platform to characterize various cellular responses in a high-throughput fashion.