On-chip CO2 control for microfluidic cell culture

Carbon dioxide partial pressure (P(CO(2))) was controlled on-chip by flowing pre-equilibrated aqueous solutions through control channels across the device. Elevated P(CO(2)) (e.g. 0.05 atm) was modulated in neighboring stagnant channels via equilibration through the highly gas permeable substrate, poly(dimethylsiloxane) (PDMS). Stable gradients in P(CO(2)) were demonstrated with a pair of control lines in a source-sink configuration. P(CO(2)) equilibration was found to be sufficiently rapid (minutes) and stable (days) to enable long-term microfluidic culture of mammalian cells. The aqueous solutions flowing through the device also mitigated pervaporative losses at sustained elevated temperatures (e.g. 37 C), as compared to flowing humidified gas through the control lines to control P(CO(2)). Since pervaporation (and the associated increase in osmolality) was minimized, stopped-flow cell culture became possible, wherein cell secretions can accumulate within the confined environment of the microfluidic culture system. This strategy was utilized to demonstrate long-term (> 7 days) microfluidic culture of mouse fibroblasts under stopped-flow conditions without requiring the microfluidic system to be placed inside a cell culture incubator.     

Rapid spatial and temporal controlled signal delivery over large cell culture areas

Controlled chemical delivery in microfluidic cell culture devices often relies on slowly evolving diffusive gradients, as the spatial and temporal control provided by fluid flow results in significant cell-perturbation. In this paper we introduce a microfluidic device architecture that allows for rapid spatial and temporal soluble signal delivery over large cell culture areas without fluid flow over the cells. In these devices the cell culture well is divided from a microfluidic channel located directly underneath the chamber by a nanoporous membrane. This configuration requires chemical signals in the microchannel to only diffuse through the thin membrane into large cell culture area, rather than diffuse in from the sides. The spatial chemical pattern within the microfluidic channel was rapidly transferred to the cell culture area with good fidelity through diffusion. The cellular temporal response to a step-function signal showed that dye reached the cell culture surface within 45 s, and achieved a static concentration in under 6 min. Chemical pulses of less than one minute were possible by temporally alternating the signal within the microfluidic channel, enabling rapid flow-free chemical microenvironment control for large cell culture areas.     

Development of Microdroplet Generation Method for Organic Solvents Used in Chemical Synthesis

Recently, chemical operations with microfluidic devices, especially droplet-based operations, have attracted considerable attention because they can provide an isolated small-volume reaction field. However, analysis of these operations has been limited mostly to aqueous-phase reactions in water droplets due to device material restrictions. In this study, we have successfully demonstrated droplet formation of five common organic solvents frequently used in chemical synthesis by using a simple silicon/glass-based microfluidic device. When an immiscible liquid with surfactant was used as the continuous phase, the organic solvent formed droplets similar to water-in-oil droplets in the device. In contrast to conventional microfluidic devices composed of resins, which are susceptible to swelling in organic solvents, the developed microfluidic device did not undergo swelling owing to the high chemical resistance of the constituent materials. Therefore, the device has potential applications for various chemical reactions involving organic solvents. Furthermore, this droplet generation device enabled control of droplet size by adjusting the liquid flow rate. The droplet generation method proposed in this work will contribute to the study of organic reactions in microdroplets and will be useful for evaluating scaling effects in various chemical reactions.     

Characterization of a membrane-based gradient generator for use in cell-signaling studies

This paper describes a method to create stable chemical gradients without requiring fluid flow. The absence of fluid flow makes this device amenable to cell signaling applications where soluble factors can impact cell behavior. This device consists of a membrane-covered source region and a large volume sink region connected by a microfluidic channel. The high fluidic resistance of the membrane limits fluid flow caused by pressure differences in the system, but allows diffusive transport of a chemical species through the membrane and into the channel. The large volume sink region at the end of the microfluidic channel helps to maintain spatial and temporal stability of the gradient. The chemical gradient in a 0.5 mm region near the sink region experiences a maximum of 10 percent change between the 6 and 24 h data points. We present the theory, design, and characterization of this device and provide an example of neutrophil chemotaxis as proof of concept for future quantitative cell-signaling applications.     

An electrochemically driven poly(dimethylsiloxane) microfluidic actuator: oxygen sensing and programmable flows and pH gradients

We describe the fabrication and performance of an integrated microelectrochemical reactor-a design possessing utility for multiple applications that include electrochemical sensing, the generation and manipulation of in-channel microfluidic pH gradients, and fluid actuation and flow. The device architecture is based on a three-electrode electrochemical cell design that incorporates a Pt interdigitated array (IDA) working (WE), a Pt counter (CE), and Ag pseudo-reference (RE) electrodes within a microfluidic network in which the WE is fully immersed in a liquid electrolyte confined in the channels. The microchannels are made from a conventional poly(dimethylsiloxane)(PDMS) elastomer, which serves also as a thin gas-permeable membrane through which gaseous reactants in the external ambient environment are supplied to the working electrode by diffusion. Due to the high permeability of oxygen through PDMS, the microfluidic cell supports significantly (>order of magnitude) higher current densities in the oxygen reduction reaction (ORR) than those measured in conventional (quiescent) electrochemical cells for the same electrode areas. We demonstrate in this work that, when operated at constant potential under mass transport control, the device can be utilized as a membrane-covered oxygen sensor, the response of which can be tuned by varying the thickness of the PDMS membrane. Depending on the experimental conditions under which the electrochemical ORR is performed, the data establish that the device can be operated as both a programmable pH gradient generator and a microfluidic pump.     

A low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami

The lab-on-a-chip concept has led to several point-of-care (POC) diagnostic microfluidic platforms. However, few of these can process raw samples for molecular diagnosis and fewer yet are suited for use in a resource-limited setting without permanent electrical infrastructure. We present here a very low cost paper microfluidic device for POC extraction of bacterial DNA from raw viscous samples--a challenge for conventional microfluidic platforms. This is an example of "microfluidic origami" in that the system is activated by folding; demonstrated here is room temperature cell lysis and DNA extraction from pig mucin (simulating sputum) spiked with E. coli without the use of external power. The microfluidic origami device features dry reagent storage and rehydration of the lysis buffer. We demonstrate DNA extraction from samples with a bacterial load as low as 33 CFU ml(-1). Extraction times, starting from the raw sample, have been optimized to about 1.5 h without the use of external power, or to within 1 h using an oven or a heater block. The fabrication of this paper microfluidic device can be translated into high volume production in the developing world without the need for a semiconductor clean room or a microfabrication facility. The sample preparation can be performed with the addition of just the sample, water, ethanol and elute buffer to the device, thus reducing chemical hazards during transport and handling.     

Flexible microfluidic devices with three-dimensional interconnected microporous walls for gas and liquid applications

This article presents a simple, low-cost method of fabrication and the applications of flexible polystyrene microfluidic devices with three-dimensional (3D) interconnected microporous walls based on treatment using a solvent/non-solvent mixture at room temperature. The complete fabrication process from device design concept to working device can be completed in less than an hour in a regular laboratory setting, without the need for expensive equipment. Microfluidic devices were used to demonstrate gas generation and absorption reactions by acidifying water with carbon dioxide (CO(2)) gas. By selectively treating the microporous structures with oxygen plasma, acidification of water by acetic acid (distilled white vinegar) perfusion was also demonstrated with the same device design.     

Characterization of a microfluidic dispensing system for localised stimulation of cellular networks

We present a 3-D microfluidic device designed for localized drug delivery to cellular networks. The device features a flow cell comprising a main channel for nutrient delivery as well as multiple channels for drug delivery. This device is one key component of a larger, fully integrated system now under development, based upon a microelectrode array (MEA) with on-chip CMOS circuitry for recording and stimulation of electrogenic cells (e.g. neurons, cardiomyocytes). As a critical system unit, the microfluidics must be carefully designed and characterized to ensure that candidate drugs are delivered to specific regions of the culture at known concentrations. Furthermore, microfluidic design and functionality is dictated by the size, geometry, and material/electrical characteristics of the CMOS MEA. Therefore, this paper reports on the design considerations and fabrication of the flow cell, including theoretical and experimental analysis of the mass transfer properties of the nutrient and drug flows, which are in good agreement with one another. To demonstrate proof of concept, the flow cell was mounted on a dummy CMOS chip, which had been plated with HL-1 cardiomyocytes. A test chemical compound was delivered to the cell culture in a spatially resolved manner. Envisioned applications of this stand-alone system include simultaneous toxicological testing of multiple compounds and chemical stimulation of natural neural networks for neuroscience investigations.     

Quantitatively controlled in situ formation of hydrogel membranes in microchannels for generation of stable chemical gradients

The in situ formation of membranes in microfluidic channels has been given attention because of their great potential in the separation of components, cell culture support for tissue engineering, and molecular transport for generation of chemical gradients. Among these, the porous membranes in microchannels are vigorously applied to generate stable chemical gradients for chemotaxis-dependent cell migration assays. Previous work on the in situ fabrication of membranes for generating the chemical gradient, however, has had several disadvantages, such as fluid leaking, uncontrollable membrane thickness, need of extra equipment, and difficulty in realizing stable interfacial layers. In this paper, we report a novel technique for the in situ formation of membranes within microchannels using enzymatically crosslinkable hydrogels and microfluidic techniques. The thickness of the membrane can be controlled quantitatively by adjusting the crosslinking reaction time and velocity of the microfluidics. By using these techniques, parallel dual hydrogel membranes were prepared within microchannels and these were used for the generation of stable concentration gradients. Moreover, the migration of Salmonella typhimurium was monitored to validate the efficacy of the chemical gradients. These results suggest that our in situ membrane system can be used as a simple platform to understand many cellular activities, including cell adhesion and migration directed by chemotaxis or complex diffusions from biological fluids in three-dimensional microstructures.     

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.     

Concentration landscape generators for shear free dynamic chemical stimulation

In this paper we first introduce a novel fabrication process, which allows for easy integration of thin track-etched nanoporous membranes, within 2D or 3D microchannel networks. In these networks, soluble chemical compounds can diffuse out of the channels through well-defined and spatially organized microfabricated porous openings. Interestingly, multiple micron-scale porous areas can be integrated in the same device and each of these areas can be connected to a different microfluidic channel and reservoir. We then present and characterize several membrane-based microdevices and their use for the generation of stable diffusible concentration gradients and complex dynamic chemical landscapes under shear free conditions. We also demonstrate how a simple flow-focusing geometry can be used to generate "on-demand" concentration profiles. In turn, these devices should provide an ideal experimental framework for high throughput cell-based assays: long term high-resolution video microscopy experiments can be performed, under multiple spatially and temporally controlled chemical conditions, with simple protocols and in a cell-friendly environment.     

Integrated ionic liquid-based electrofluidic circuits for pressure sensing within polydimethylsiloxane microfluidic systems

This paper reports a novel pressure sensor with an electrical readout based on electrofluidic circuits constructed by ionic liquid (IL)-filled microfluidic channels. The developed pressure sensor can be seamlessly fabricated into polydimethylsiloxane (PDMS) microfluidic systems using the well-developed multilayer soft lithography (MSL) technique without additional assembly or sophisticated cleanroom microfabrication processes. Therefore, the device can be easily scaled up and is fully disposable. The pressure sensing is achieved by measuring the pressure-induced electrical resistance variation of the constructed electrofluidic resistor. In addition, an electrofluidic Wheatstone bridge circuit is designed for accurate and stable resistance measurements. The pressure sensor is characterized using pressurized nitrogen gas and various liquids which flow into the microfluidic channels. The experimental results demonstrate the great long-term stability (more than a week), temperature stability (up to 100 °C), and linear characteristics of the developed pressure sensing scheme. Consequently, the integrated microfluidic pressure sensor developed in this paper is promising for better monitoring and for characterizing the flow conditions and liquid properties inside the PDMS microfluidic systems in an easier manner for various lab on a chip applications.     

Three-dimensional interconnected microporous poly(dimethylsiloxane) microfluidic devices

This technical note presents a fabrication method and applications of three-dimensional (3D) interconnected microporous poly(dimethylsiloxane) (PDMS) microfluidic devices. Based on soft lithography, the microporous PDMS microfluidic devices were fabricated by molding a mixture of PDMS pre-polymer and sugar particles in a microstructured mold. After curing and demolding, the sugar particles were dissolved and washed away from the microstructured PDMS replica revealing 3D interconnected microporous structures. Other than introducing microporous structures into the PDMS replica, different sizes of sugar particles can be used to alter the surface wettability of the microporous PDMS replica. Oxygen plasma assisted bonding was used to enclose the microstructured microporous PDMS replica using a non-porous PDMS with inlet and outlet holes. A gas absorption reaction using carbon dioxide (CO(2)) gas acidified water was used to demonstrate the advantages and potential applications of the microporous PDMS microfluidic devices. We demonstrated that the acidification rate in the microporous PDMS microfluidic device was approximately 10 times faster than the non-porous PDMS microfluidic device under similar experimental conditions. The microporous PDMS microfluidic devices can also be used in cell culture applications where gas perfusion can improve cell survival and functions.     

Perfusion-based microfluidic device for three-dimensional dynamic primary human hepatocyte cell culture in the absence of biological or synthetic matrices or coagulants

We describe a perfusion-based microfluidic device for three-dimensional (3D) dynamic primary human hepatocyte cell culture. The microfluidic device was used to promote and maintain 3D tissue-like cellular morphology and cell-specific functionality of primary human hepatocytes by restoring membrane polarity and hepatocyte transport function in vitro without the addition of biological or synthetic matrices or coagulants. A unique feature of our dynamic cell culture device is the creation of a microenvironment, without the addition of biological or synthetic matrices or coagulants, that promotes the 3D organization of hepatocytes into cord-like structures that exhibit functional membrane polarity as evidenced by the expression of gap junctions and the formation of an extended, functionally active, bile canalicular network.     

Optimization of a permeation-based microfluidic direct formic acid fuel cell (DFAFC)

A design for a passive, air-breathing microfluidic fuel cell utilizing formic acid (FA) as a fuel is described and its performance characterized. The fuel cell integrates high surface area platinum (cathode) and palladium-platinum (anode) alloy electrodes within a PDMS microfluidic network that keeps them fully immersed in a liquid electrolyte. The polymer network that comprises the device also serves as a self-supporting membrane through which FA and oxygen are supplied to the alloy anode and cathode, respectively, by passive permeation from external sources. The cell is based on a planar form-factor and in its operation exploits FA concentration gradients that form across the PDMS membrane. These latter gradients allow the device to operate stably, producing a nearly constant limiting power density of ~0.2 mW/cm2, without driven laminar flow of fluids or the incorporation of an in-channel separator between the anodic and the cathodic compartments. The power output of this elementary device in air is subject to electrolyte mass transport impacts, which can be reduced for a given design rule by decreasing the internal ohmic resistance of the cell. The results suggest that operational stability can be improved by decreasing the kinetic losses imposed on the cathode side of the cell due to FA crossover and modalities for doing so, such as by increasing the efficiency of fuel capture at the anode.     

Microfluidic device generating stable concentration gradients for long term cell culture: application to Wnt3a regulation of β-catenin signaling

In developing tissues, proteins and signaling molecules present themselves in the form of concentration gradients, which determine the fate specification and behavior of the sensing cells. To mimic these conditions in vitro, we developed a microfluidic device designed to generate stable concentration gradients at low hydrodynamic shear and allowing long term culture of adhering cells. The gradient forms in a culture space between two parallel laminar flow streams of culture medium at two different concentrations of a given morphogen. The exact algorithm for defining the concentration gradients was established with the aid of mathematical modeling of flow and mass transport. Wnt3a regulation of β-catenin signaling was chosen as a case study. The highly conserved Wnt-activated β-catenin pathway plays major roles in embryonic development, stem cell proliferation and differentiation. Wnt3a stimulates the activity of β-catenin pathway, leading to translocation of β-catenin to the nucleus where it activates a series of target genes. We cultured A375 cells stably expressing a Wnt/β-catenin reporter driving the expression of Venus, pBARVS, inside the microfluidic device. The extent to which the β-catenin pathway was activated in response to a gradient of Wnt3a was assessed in real time using the BARVS reporter gene. On a single cell level, the β-catenin signaling was proportionate to the concentration gradient of Wnt3a; we thus propose that the modulation of Wnt3a gradients in real time can provide new insights into the dynamics of β-catenin pathway, under conditions that replicate some aspects of the actual cell-tissue milieu. Our device thus offers a highly controllable platform for exploring the effects of concentration gradients on cultured cells.     

A software-programmable microfluidic device for automated biology

Specific-purpose microfluidic devices have had considerable impact on the biological and chemical sciences, yet their use has largely remained limited to specialized laboratories. Here we present a general-purpose software-programmable microfluidic device which is capable of performing a multitude of low- and high-level functions without requiring any hardware modifications. To demonstrate the applicability and modularity of the device we implemented a variety of applications such as a microfluidic display, fluid metering and active mixing, surface immunoassays, and cell culture. We believe that analogously to personal computers, programmable, general-purpose devices will increase the accessibility and advance the pervasiveness of microfluidic technology.     

Mucin (MUC5AC) expression by lung epithelial cells cultured in a microfluidic gradient device

We have developed a microfluidic gradient device for controlling mucin gene expression of NCI-H292 epithelial cells derived from lung tissues. We hypothesized that gradient profiles would control mucin gene expression of lung epithelial cells. However, it was not possible to generate various stable gradient profiles using conventional culture methods. To address this limitation, we used a microfluidic gradient device to create various gradient profiles (i.e. non-linear, linear, and flat) in a temporal and spatial manner. NCI-H292 lung epithelial cells were exposed to concentration gradients of epidermal growth factor in a microfluidic gradient device with continuous medium perfusion. We demonstrated an effect of gradient profiles on mucin expression of lung epithelial cells cultured in the microfluidic gradient device. It was revealed that NCI-H292 lung epithelial cells exposed to the flat gradient profile of the epidermal growth factor exhibited high expression of mucin as compared with cells exposed to non-linear and linear gradient profiles. Therefore, this microfluidic gradient device could be a potentially useful tool for regulating the mucin expression of lung epithelial cells exposed to chemokine gradient profiles.     

A micropillar-integrated smart microfluidic device for specific capture and sorting of cells

An integrated smart microfluidic device consisting of nickel micropillars, microvalves, and microchannels was developed for specific capture and sorting of cells. A regular hexagonal array of nickel micropillars was integrated on the bottom of a microchannel by standard photolithography, which can generate strong induced magnetic field gradients under an external magnetic field to efficiently trap superparamagnetic beads (SPMBs) in a flowing stream, forming a bed with sufficient magnetic beads as a capture zone. Fluids could be manipulated by programmed controlling the integrated air-pressure-actuated microvalves, based on which in situ bio-functionalization of SPMBs trapped in the capture zone was realized by covalent attachment of specific proteins directly to their surface on the integrated microfluidic device. In this case, only small volumes of protein solutions (62.5 nL in the capture zone; 375 nL in total volume needed to fill the device from inlet A to the intersection of outlet channels F and G) can meet the need for protein! The newly designed microfluidic device reduced greatly chemical and biological reagent consumption and simplified drastically tedious manual handling. Based on the specific interaction between wheat germ agglutinin (WGA) and N-acetylglucosamine on the cell membrane, A549 cancer cells were effectively captured and sorted on the microfluidic device. Capture efficiency ranged from 62 to 74%. The integrated microfluidic device provides a reliable technique for cell sorting.     

Microfabricated devices for biomolecule encapsulation

Biomolecule encapsulation in droplets is important for miniaturizing biological assays to reduce reagent consumption, cost and time of analysis, and can be most effectively achieved by using microfabricated devices. Microfabricated fluidic devices can generate emulsified drops of uniform size with controlled dimensions and contents. Biological and chemical components such as cells, microgels, beads, hydrogel precursors, polymer initiators, and other droplets can be encapsulated within these drops. Encapsulated emulsions are appealing for a variety of applications since drops can be used as tiny reaction vessels to perform high-throughput reactions at fast rates, consuming minimal sample and solvent amounts due to the small size (micron diameters) of the emulsion drops. Facile mixing and droplet coalescence allow for a diversity of assays to be performed on-chip with tunable parameters. The simplicity of operation and speed of analysis with microencapsulated drops lends itself well to an array of quantitative biomolecular studies such as directed evolution, single-molecule DNA amplification, single-cell encapsulation, high-throughput sequencing, enzyme kinetics, and microfluidic cell culture. This review highlights recent advances in the field of microfabricated encapsulating devices, emphasizing the development of emulsifying encapsulations, device design, and current assays that are performed using encapsulating droplets.