Gelatin based microfluidic devices for cell culture

We have developed a technique for fabricating microfluidic devices from gelatin using a natural crosslinking process. Gelatin, crosslinked with the naturally occurring enzyme transglutaminase is molded to produce microchannels suitable for adherent cell culture and analysis. The autofluorescence of the material was shown to be minimal and within the range of typical background, ensuring utility with analyses using fluorescent dyes and labels would not be affected. Also, normal murine mammary epithelial cells were successfully cultured in the microchannels. The morphology of these adherent epithelial cells was shown to be significantly different for cells grown on rigid tissue culture plastic in either macro- or microscale cultures (even in the presence of a surface coating of gelatin) than those grown on the flexible crosslinked gelatin microchannels. Using these devices, the effects of both the extracellular matrix and soluble factors on cellular behavior and differentiation can be studied in microenvironments that more closely mimic the in vivo environment.     

Patterned cell culture inside microfluidic devices

This paper describes a simple plasma-based dry etching method that enables patterned cell culture inside microfluidic devices by allowing patterning, fluidic bonding and sterilization steps to be carried out in a single step. This plasma-based dry etching method was used to pattern cell-adhesive and non-adhesive areas on the glass and polystyrene substrates. The patterned substrate was used for selective attachment and growth of human umbilical vein endothelial cells, MDA-MB-231 human breast cancer cells, NIH 3T3 mouse fibroblasts, and primary rat cortical neurons. Finally, we have successfully combined the dry-patterned substrate with a microfluidic device. Patterned primary rat neurons were maintained for up to 6 days inside the microfluidic devices and the neurons' somas and processes were confined to the cell-adhesive region. The method developed in this work offers a convenient way of micropatterning biomaterials for selective attachment of cells on the substrates, and enables culturing of patterned cells inside microfluidic devices for a number of biological research applications where cells need to be exposed to well-controlled fluidic microenvironment.     

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.     

Polyurethane-based microfluidic devices for blood contacting applications

Protein adsorption on PDMS surfaces poses a significant challenge in microfluidic devices that come into contact with biofluids such as blood. Polyurethane (PU) is often used for the construction of medical devices, but despite having several attractive properties for biointerfacing, it has not been widely used in microfluidic devices. In this work we developed two new fabrication processes for making thin, transparent and flexible PU-based microfluidic devices. Methods for the fabrication and bonding of microchannels, the integration of fluidic interconnections and surface modification with hydrophilic polyethylene oxide (PEO) to reduce protein adsorption are detailed. Using these processes, microchannels were produced having high transparency (96% that of glass in visible light), high bond strength (326.4 kPa) and low protein adsorption (80% reduction in fibrinogen adsorption vs. unmodified PDMS), which is critical for prevention of fouling. Our findings indicate that PEO modified PU could serve as an effective alternative to PDMS in blood contacting microfluidic applications.     

Micro- and nanometer-scale patterned surface in a microchannel for cell culture in microfluidic devices

A novel microdevice which had a micro- and nanometer-scale patterned surface for cell adhesion in a microchip was developed. The surface had a metal pattern fabricated by electron-beam lithography and metal sputtering and a chemical pattern consisting of a self-assembled monolayer of alkanethiol. The metal patterned surface had a gold stripe pattern which was as small as 300 nm wide and 150 nm high and both topography and chemical properties could be controlled. Mouse fibroblast NIH/3T3 cells were cultured on the patterned surface and elongated along the gold stripes. These cells recognized the size of the pattern and the chemical properties on the pattern though it was much smaller than they were. There was satisfactory cell growth under fresh medium flow in the microchip. The combination of the patterned surface and the microchip provides cells with a novel environment for their growth and will facilitate many cellular experiments. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Perforated membrane method for fabricating three-dimensional polydimethylsiloxane microfluidic devices

A procedure is described for making layer-to-layer interconnections in polydimethylsiloxane (PDMS) microfluidic devices. Thin ( approximately 50 mum) perforated PDMS membranes are bonded to thicker (0.1 cm or more) PDMS slabs by means of thermally cured PDMS prepolymer to form a three-dimensional (3D) channel structure, which may contain channel or valve arrays that can pass over and under one another. Devices containing as many as two slabs and three perforated membranes are demonstrated. We also present 3D PDMS microfluidic devices for display and for liquid dispensing.     

High throughput method for prototyping three-dimensional, paper-based microfluidic devices

This paper describes an efficient and high throughput method for fabricating three-dimensional (3D) paper-based microfluidic devices. The method avoids tedious alignment and assembly steps and eliminates a major bottleneck that has hindered the development of these types of devices. A single researcher now can prepare hundreds of devices within 1 h.     

Generation of oxygen gradients in microfluidic devices for cell culture using spatially confined chemical reactions

This paper reports a microfluidic device capable of generating oxygen gradients for cell culture using spatially confined chemical reactions with minimal chemical consumption. The microfluidic cell culture device is constructed by single-layer polydimethylsiloxane (PDMS) microfluidic channels, in which the cells can be easily observed by microscopes. The device can control the oxygen gradients without the utilization of bulky pressurized gas cylinders, direct addition of oxygen scavenging agents, or tedious gas interconnections and sophisticated flow control. In addition, due to the efficient transportation of oxygen within the device using the spatially confined chemical reactions, the microfluidic cell culture device can be directly used in conventional cell incubators without altering their gaseous compositions. The oxygen gradients generated in the device are numerically simulated and experimentally characterized using an oxygen-sensitive fluorescence dye. In this paper, carcinomic human alveolar basal epithelial (A549) cells have been cultured in the microfluidic device with a growth medium and an anti-cancer drug (Tirapazamine, TPZ) under various oxygen gradients. The cell experiment results successfully demonstrate the hyperoxia-induced cell death and hypoxia-induced cytotoxicity of TPZ. In addition, the results confirm the great cell compatibility and stable oxygen gradient generation of the developed device. Consequently, the microfluidic cell culture device developed in this paper is promising to be exploited in biological labs with minimal instrumentation to study cellular responses under various oxygen gradients.     

Lamination-based rapid prototyping of microfluidic devices using flexible thermoplastic substrates

Transposing highly sensitive DNA separation methods (such as DNA sequencing with high read length or the detection of point mutations) to microchip format without loss of resolution requires fabrication of relatively long (approx. 10 cm) microchannels along with sharp injection bands. Conventional soft lithography methods, such as mold casting or hot-embossing in a press, are not convenient for fabricating long channels. We have developed a lamination-based replication technique for rapid fabrication of sealed microfluidic devices with a 10 cm long, linear separation channel. These devices are fabricated in thin cyclo-olefin copolymer (COC) plastic substrates, thus making the device flexible and capable of assuming a range of 3-D configurations. Due to the good optical properties of COC, this new family of devices combines multiple advantages of planar microfluidics and fused-silica capillaries.     

Cell immersion and cell dipping in microfluidic devices

Combining deflective dielectrophoretic barriers with controlled pressure driven liquid flows in microfluidic devices allows accurate handling of particles such as biological cells in suspensions. Working towards cell-based lab-on-a-chip applications, a platform permitting rapid testing of devices having different dielectrophoretic and fluidic subunits was developed. The performance of such a system is shown in the cases of (A) flooding a small number of immobilised cells with a dye and (B) transient buffer swapping of a large number of cells in flow. The transition times for moving cells from one reagent to the other are below 0.5 s in the case of flow-through cell dipping.     

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.     

In-channel atom-transfer radical polymerization of thermoset polyester microfluidic devices for bioanalytical applications

A new technique for polymer microchannel surface modification, called in-channel atom-transfer radical polymerization, has been developed and applied in the surface derivatization of thermoset polyester (TPE) microdevices with poly(ethylene glycol) (PEG). X-ray photoelectron spectroscopy, electroosmotic flow (EOF), and contact angle measurements indicate that PEG has been grafted on the TPE surface. Moreover, PEG-modified microchannels have much lower and more pH-stable EOF, more hydrophilic surfaces and reduced nonspecific protein adsorption. Capillary electrophoresis separation of amino acid and peptide mixtures in these PEG-modified TPE microchips had good reproducibility. Phosducin-like protein and phosphorylated phosducin-like protein were also separated to measure the phosphorylation efficiency. Our results indicate that PEG-grafted TPE microchips have broad potential application in biomolecular analysis.     

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.     

Recent applications of AC electrokinetics in biomolecular analysis on microfluidic devices

AC electrokinetics is a generic term that refers to an induced motion of particles and fluids under nonuniform AC electric fields. The AC electric fields are formed by application of AC voltages to microelectrodes, which can be easily integrated into microfluidic devices by standard microfabrication techniques. Moreover, the magnitude of the motion is large enough to control the mass transfer on the devices. These advantages are attractive for biomolecular analysis on the microfluidic devices, in which the characteristics of small space and microfluidics have been mainly employed. In this review, I describe recent applications of AC electrokinetics in biomolecular analysis on microfluidic devices. The applications include fluid pumping and mixing by AC electrokinetic flow, and manipulation of biomolecules such as DNA and proteins by various AC electrokinetic techniques. Future prospects for highly functional biomolecular analysis on microfluidic devices with the aid of AC electrokinetics are also discussed.     

Nanowire-integrated microfluidic devices for facile and reagent-free mechanical cell lysis

Cell lysis is an essential task for the detection of intracellular components. In this work, we introduce novel microfluidic devices integrated with patterned one-dimensional nanostructure arrays for facile and high-throughput mechanical cell lysis. The geometry of the hydrothermally grown ZnO nanowires, characterised by sharp tips and high aspect ratios, aids in anchoring the cell and tearing the plasma membrane, enabling simple and highly efficient extraction of cellular proteins and nucleic acids. This method lyses cells more effectively than conventional chemical lysis methods with simpler equipment and a shorter processing time.     

Thermoplastic microfluidic devices and their applications in protein and DNA analysis

Microfluidics is a platform technology that has been used for genomics, proteomics, chemical synthesis, environment monitoring, cellular studies, and other applications. The fabrication materials of microfluidic devices have traditionally included silicon and glass, but plastics have gained increasing attention in the past few years. We focus this review on thermoplastic microfluidic devices and their applications in protein and DNA analysis. We outline the device design and fabrication methods, followed by discussion on the strategies of surface treatment. We then concentrate on several significant advancements in applying thermoplastic microfluidic devices to protein separation, immunoassays, and DNA analysis. Comparison among numerous efforts, as well as the discussion on the challenges and innovation associated with detection, is presented.     

Macro-to-micro interfaces for microfluidic devices

Since the concept of miniaturized total analysis systems (microTAS) was invented, a great number of microfluidic devices have been demonstrated for a variety of applications. However, an important hurdle that still needs to be cleared is the connection of a microfluidic device with the rest of the world, which is often referred to as the macro-to-micro interface, interconnect, or world-to-chip interface. In this review, we will examine the methods used by pioneers in the field and other investigators, review the approaches for capillary electrophoresis-based devices and those using pneumatic pumping, and present additional discussion on interface standardization and choosing and designing interconnects for your applications.     

Bioanalysis in microfluidic devices

Microfabricated bioanalytical devices (also referred to as laboratory-on-a-chip or micro-TAS) offer highly efficient platforms for simultaneous analysis of a large number of biologically important molecules, possessing great potential for genome, proteome and metabolome studies. Development and implementation of microfluidic-based bioanalytical tools involves both established and evolving technologies, including microlithography, micromachining, micro-electromechanical systems technology and nanotechnology. This article provides an overview of the latest developments in the key device subject areas and the basic interdisciplinary technologies. Important aspects of DNA and protein analysis, interfacing issues and system integration are all thoroughly discussed, along with applications for this novel "synergized" technology in high-throughput separations of biologically important molecules. This review also gives a better understanding of how to utilize these technologies as well as to provide appropriate technical solutions to problems perceived as being more fundamental.     

Monolithic media in microfluidic devices for proteomics

Considerable effort has been invested in the development of integrated microfluidic devices for fast and highly efficient proteomic studies. Among various fabrication techniques for the preparation of analytical components (separation columns, reactors, extractors, valves, etc.) in integrated microchips, in situ fabrication of monolithic media is receiving increasing attention. This is mainly due to the ease and simplicity of preparation of monolithic media and the availability of various precursors and chemistries. In addition, UV-initiated photopolymerization technique enables the incorporation of multiple analytical components into specified parts of a single microchip using photomasks. This review summarizes preparation methods for monolithic media and their application as microfluidic analytical components in microchips.     

Design, fabrication and implementation of a novel multi-parameter control microfluidic platform for three-dimensional cell culture and real-time imaging

New and more biologically relevant in vitro models are needed for use in drug development, regenerative medicine, and fundamental scientific investigation. While the importance of the extracellular microenvironment is clear, the ability to investigate the effects of physiologically relevant biophysical and biochemical factors is restricted in traditional cell culture platforms. Moreover, the versatility for multi-parameter manipulation, on a single platform, with the optical resolution to monitor the dynamics of individual cells or small population is lacking. Here we introduce a microfluidic platform for 3D cell culture in biologically derived or synthetic hydrogels with the capability to monitor cellular dynamics in response to changes in their microenvironment. Direct scaffold microinjection, was employed to incorporate 3D matrices into microfluidic devices. Our system geometry permits a unique window for studying directional migration, e.g. sprouting angiogenesis, since sprouts grow predominantly in the microscopic viewing plane. In this study, we demonstrate the ability to generate gradients (non-reactive solute), surface shear, interstitial flow, and image cells in situ. Three different capillary morphogenesis assays are demonstrated. Human adult dermal microvascular endothelial cells (HMVEC-ad) were maintained in culture for up to 7 days during which they formed open lumen-like structures which was confirmed with confocal microscopy and by perfusion with fluorescent microspheres. In the sprouting assay, time-lapse movies revealed cellular mechanisms and dynamics (filopodial projection/retraction, directional migration, cell division and lumen formation) during tip-cell invasion of underlying 3D matrix and subsequent lumen formation.