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.     

集成药物代谢微流控芯片的研制

本文研制了一种集成药物代谢微流控芯片, 此芯片可以同时完成药物代谢物的分子检测和代谢过程对药物细胞毒性的影响评价, 为进一步的药物代谢和药物相互作用研究奠定了良好的基础.     

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.     

Cell docking inside microwells within reversibly sealed microfluidic channels for fabricating multiphenotype cell arrays

We present a soft lithographic method to fabricate multiphenotype cell arrays by capturing cells within an array of reversibly sealed microfluidic channels. The technique uses reversible sealing of elastomeric polydimethylsiloxane (PDMS) molds on surfaces to sequentially deliver various fluids or cells onto specific locations on a substrate. Microwells on the substrate were used to capture and immobilize cells within low shear stress regions inside channels. By using an array of channels it was possible to deposit multiple cell types, such as hepatocytes, fibroblasts, and embryonic stem cells, on the substrates. Upon formation of the cell arrays on the substrate, the PDMS mold could be removed, generating a multiphenotype array of cells. In addition, the orthogonal alignment and subsequent attachment of a secondary array of channels on the patterned substrates could be used to deliver fluids to the patterned cells. The ability to position many cell types on particular regions within a two dimensional substrate could potentially lead to improved high-throughput methods applicable to drug screening and tissue engineering.     

Integrated microfluidic array plate (iMAP) for cellular and molecular analysis

Just as the Petri dish has been invaluable to the evolution of biomedical science in the last 100 years, microfluidic cell assay platforms have the potential to change significantly the way modern biology and clinical science are performed. However, an evolutionary process of creating an efficient microfluidic array for many different bioassays is necessary. Specifically for a complete view of a cell response it is essential to incorporate cytotoxic, protein and gene analysis on a single system. Here we present a novel cellular and molecular analysis platform, which allows access to gene expression, protein immunoassay, and cytotoxicity information in parallel. It is realized by an integrated microfluidic array plate (iMAP). The iMAP enables sample processing of cells, perfusion based cell culture, effective perturbation of biologic molecules or drugs, and simultaneous, real-time optical analysis for different bioassays. The key features of the iMAP design are the interface of on-board gravity driven flow, the open access input fluid exchange and the highly efficient sedimentation based cell capture mechanism (～100% capture rates). The operation of the device is straightforward (tube and pump free) and capable of handling dilute samples (5-cells per experiment), low reagent volumes (50 nL per reaction), and performing single cell protein and gene expression measurements. We believe that the unique low cell number and triple analysis capabilities of the iMAP platform can enable novel dynamic studies of scarce cells.     

Integrated microelectrode array and microfluidics for temperature clamp of sensory neurons in culture

A device for cell culture is presented that combines MEMS technology and liquid-phase photolithography to create a microfluidic chip that influences and records electrical cellular activity. A photopolymer channel network is formed on top of a multichannel microelectrode array. Preliminary results indicated successful local thermal control within microfluidic channels and control of lamina position over the electrode array. To demonstrate the biological application of such a device, adult dissociated dorsal root ganglion neurons with a subpopulation of thermally-sensitive cells are attached onto the electrode array. Using laminar flow, dynamic control of local temperature of the neural cells was achieved while maintaining a constant chemical culture medium. Recording the expected altered cellular activity confirms the success of the integrated device.     

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.     

Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves

Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.     

A microfluidic platform for high-sensitivity, real-time drug screening on C. elegans and parasitic nematodes

This paper describes a new microfluidic platform for screening drugs and their dose response on the locomotion behavior of free living nematodes and parasitic nematodes. The system offers a higher sensitivity drug screening chip which employs a combination of existing and newly developed methods. Real-time observation of the entire drug application process (i.e. the innate pre-exposure locomotion, the transient response during drug exposure and the time-resolved, post-exposure behavior) at a single worm resolution is made possible. The chip enables the monitoring of four nematode parameters (number of worms responsive, number of worms leaving the drug well, average worm velocity and time until unresponsiveness). Each parameter generates an inherently different dose response; allowing for a higher resolution when screening for resistance. We expect this worm chip could be used as a robust cross species, cross drug platform. Existing nematode motility and migration assays do not offer this level of sophistication. The device comprises two principal components: behavioral microchannels to study nematode motility and a drug well for administering the dose and observing drug effects as a function of exposure time. The drug screening experiment can be described by three main steps: (i) 'pre-exposure study'- worms are inserted into the behavioral channels and their locomotion is characterized, (ii) 'dose exposure'- worms are guided from the behavioral microchannels into the drug well and held for a predefined time, during which time their transient response to the dose is characterized and (iii) 'post-exposure study'- worms are guided back into the behavioral microchannels where their locomotion (i.e. their time-resolved response to the dose) is characterized and compared to pre-exposure motility. The direction of nematodes' movement is reliably controlled by the application of an electric field within a defined range. Control experiments (e.g. in the absence of any drug) confirm that the applied electric fields do not affect the worms' motility or viability. We demonstrate the workability of the microfluidic platform on free living Caenorhabditis elegans (wild-type N2 and levamisole resistant ZZ15 lev-8) and parasitic Oesophagotomum dentatum (levamisole-sensitive, SENS and levamisole-resistant, LEVR) using levamisole (a well-studied anthelmintic) as the test drug. The proposed scheme of drug screening on a microfluidic device is expected to significantly improve the resolution, sensitivity and data throughput of in vivo testing, while offering new details on the transient and time-resolved exposure effects of new and existing anthelmintics.     

A novel 3D mammalian cell perfusion-culture system in microfluidic channels

Mammalian cells cultured on 2D surfaces in microfluidic channels are increasingly used in drug development and biological research applications. These systems would have more biological or clinical relevance if the cells exhibit 3D phenotypes similar to the cells in vivo. We have developed a microfluidic channel based system that allows cells to be perfusion-cultured in 3D by supporting them with adequate 3D cell-cell and cell-matrix interactions. The maximal cell-cell interaction was achieved by perfusion-seeding cells through an array of micropillars; and 3D cell-matrix interactions were achieved by a polyelectrolyte complex coacervation process to form a thin layer of matrix conforming to the 3D cell shapes. Carcinoma cell lines (HepG2, MCF7), primary differentiated (hepatocytes) and primary progenitor cells (bone marrow mesenchymal stem cells) were perfusion-cultured for 72 hours to 1 week in the microfluidic channel, which preserved their 3D cyto-architecture and cell-specific functions or differentiation competence. This transparent 3D microfluidic channel-based cell culture system also allows direct optical monitoring of cellular events for a wide range of applications.     

An integrated microfluidic device for two-dimensional combinatorial dilution

High-throughput preparation of multi-component solutions is an integral process in biology, chemistry and materials science for screening, diagnostics and analysis. Compact microfluidic systems enable such processing with low reagent volumes and rapid testing. Here we present a microfluidic device that incorporates two gradient generators, a tree-like generator and a new microfluidic active injection system, interfaced by intermediate solution reservoirs to generate diluted combinations of input solutions within an 8 × 8 or 10 × 10 array of isolated test chambers. Three input solutions were fed into the device, two to the tree-like gradient generator and one to pre-fill the test chamber array. The relative concentrations of these three input solutions in the test chambers completely characterized device behaviour and were controlled by the number of injection cycles and the flow rate. Device behaviour was modelled by computational fluid dynamics simulations and an approximate analytic formula. The device may be used for two-dimensional (2D) combinatorial dilution by adding two solutions in different relative concentrations to each of its three inputs. By appropriate choice of the two-component input solutions, test chamber concentrations that span any triangle in 2D concentration space may be obtained. In particular, explicit inputs are given for a coarse screening of a large region in concentration space followed by a more refined screening of a smaller region, including alternate inputs that span the same concentration region but with different distributions. The ability to probe arbitrary subspaces of concentration space and to control the distribution of discrete test points within those subspaces makes the device of potential benefit for high-throughput cell biology studies and drug screening.     

Cell-based high content screening using an integrated microfluidic device

High content screening (HCS) has quickly established itself as a core technique in the early stage of drug discovery for secondary compound screening. It allows several independent cellular parameters to be measured in a single cell or populations of cells in a single assay. In this work, we describe high content screening for the multiparametric measurement of cellular responses in human liver carcinoma (HepG2) cells using an integrated microfluidic device. This device consists of multiple drug gradient generators and parallel cell culture chambers, in which the processes of liquid dilution and diffusion, micro-scale cell culture, cell stimulation and cell labeling can be integrated into a single device. The simple assay provides multiparametric measurements of plasma membrane permeability, nuclear size, mitochondrial transmembrane potential and intracellular redox states in anti-cancer drug-induced apoptosis of HepG2 cells. The established platform is able to rapidly extract the maximum of information from tumor cells in response to several drugs varying in concentration, with minimal sample and less time, which is very useful for basic biomedical research and cancer treatment.     

Influence of channel position on sample confinement in two-dimensional planar microfluidic devices

We report enhanced sample confinement on microfluidic devices using a combination of electrokinetic flow from adjacent control channels and electric field shaping with an array of channels perpendicular to the sample stream. The basic device design consisted of a single first dimension (1D) channel, intersecting an array of 32 or 96 parallel second dimension (2D) channels. To minimize sample dispersion and leakage into the parallel channels as the sample traversed the sample transfer region, control channels were placed to the left and right of the 1D and waste channels. The electrokinetic flow from the control channels confined the sample stream and acted as a buffer between the sample stream and the 2D channels. To further enhance sample confinement, the electric field was shaped parallel to the sample stream by placing the channel array in close proximity to the sample transfer region. Using COMSOL Multiphysics, initial work focused on simulating the electric fields and fluid flows in various device geometries, and the results guided device design. Following the design phase, we fabricated devices with 40, 80, and 120 microm wide control channels and evaluated the sample stream width as a function of the electric field strength ratio in the control and 1D channels (E(C)/E(1D)). For the 32 channel design, the 40 and 80 microm wide control channels produced the most effective sample confinement with stream widths as narrow as 75 microm, and for the 96 channel design, all three control channel widths generated comparable sample stream widths. Comparison of the 32 and 96 channel designs showed sample confinement scaled easily with the length of the sample transfer region.     

A cell-laden microfluidic hydrogel

The encapsulation of mammalian cells within the bulk material of microfluidic channels may be beneficial for applications ranging from tissue engineering to cell-based diagnostic assays. In this work, we present a technique for fabricating microfluidic channels from cell-laden agarose hydrogels. Using standard soft lithographic techniques, molten agarose was molded against a SU-8 patterned silicon wafer. To generate sealed and water-tight microfluidic channels, the surface of the molded agarose was heated at 71 degrees C for 3 s and sealed to another surface-heated slab of agarose. Channels of different dimensions were generated and it was shown that agarose, though highly porous, is a suitable material for performing microfluidics. Cells embedded within the microfluidic molds were well distributed and media pumped through the channels allowed the exchange of nutrients and waste products. While most cells were found to be viable upon initial device fabrication, only those cells near the microfluidic channels remained viable after 3 days, demonstrating the importance of a perfused network of microchannels for delivering nutrients and oxygen to maintain cell viability in large hydrogels. Further development of this technique may lead to the generation of biomimetic synthetic vasculature for tissue engineering, diagnostics, and drug screening applications.     

Denaturing gradient-based two-dimensional gene mutation scanning in a polymer microfluidic network

An integrated two-dimensional (2-D) DNA separation platform, combining standard gel electrophoresis with temperature gradient gel electrophoresis (TGGE) on a polymer microfluidic chip, is reported. Rather than sequentially sampling DNA fragments eluted from standard gel electrophoresis, size-resolved fragments are simultaneously electrokinetically transferred into an array of orthogonal microchannels and screened for the presence of sequence heterogeneity by TGGE in a parallel and high throughput format. A bulk heater assembly is designed and employed to externally generate a temporal temperature gradient along an array of TGGE channels. Extensive finite element modeling is performed to determine the optimal geometries of the microfluidic network for minimizing analyte band dispersion caused by interconnected channels in the network. A pH-mediated on-chip analyte stacking strategy is employed prior to the parallel TGGE separations to further reduce additional band broadening acquired during the electrokinetic transfer of DNA fragments between the first and second separation dimensions. A comprehensive 2-D DNA separation is completed in less than 5 min for positive detection of single-nucleotide polymorphisms in multiplex PCR products that vary in size and sequence.     

Chemiluminescence response of murine macrophages on multilayer microfluidic chips

We have demonstrated an integrated platform for microfluidics and chemiluminescence (CL) detection that is capable of parallel cell culture, convenient liquid manipulation, and sensitive chemiluminescent detection. Luminol-dependent CL responses induced by three different stimuli, phytohemagglutinin (PHA), concanavalin A (ConA), and lipopolysaccharides (LPS), which can evoke a CL response in macrophages, were evaluated on this microfluidic chip. We studied the dose-dependent effect of these three stimuli on CL response in murine macrophages. PHA produced the highest CL response compared to LPS and ConA. The CL intensity produced by PHA was 6.85 and four times higher than that by LPS and ConA, respectively, at the low concentration of 100 μg/ml. We have found microfluidic based CL to be a very useful screening tool, which is less laborious and more sensitive. This microfluidic system is disposable and capable of rapid device prototyping; it may prove to be very useful in clinical and pharmaceutical applications.     

Design of parallel microfluidic gradient-generating networks for studying cellular response to chemical stimuli

A microfluidic chip featuring laminar flow-based parallel gradient-generating networks was designed and fabricated. The microchip contains 5 gradient generators and 30 cell chambers where the resulting concentration gradients of drugs are delivered to stimulate on-chip cultured cells. The microfluidics exploits the advantage of lab-on-a-chip technology by integrating the generation of drug concentration gradients and a series of cell operations including seeding, culture, stimulation and staining into a chip. The microfluidic network was patterned on a glass wafer, which was further bonded to a PDMS film. A series of weir structures were fabricated on the cell culture reservoir to facilitate cell positioning and seeding. Cell injection and fluid delivery were controlled by a syringe pump. Steady parallel concentration gradients were generated by flowing two fluids in each network. Over time observation shows that the microchip was suitable for cell seeding and culture. The microchip described above was applied in studying the role of reduced glutathione (GSH) in mediating chemotherapy sensitivity of MCF-7 cells. MCF-7 cells were treated with concentration gradients of As₂O₃ and N-acetyl cysteine (NAC) for GSH modulation, followed by exposure to adriamycin. GSH levels were down-regulated upon As₂O₃ treatment and up-regulated upon NAC treatment. Suppression of intracellular GSH by treatment with As₂O₃ has been shown to increase sensitivity to adriamycin. Conversely, elevation of intracellular GSH by treatment with NAC leads to increased drug resistance. The integrated microfluidic chip is able to perform multiparametric pharmacological profiling with easy operation, and thus holds great potential for extrapolation to the cell based high-content drug screening. __________ Translated from Chinese Journal of Analytical Chemistry, 2008, 36(2): 143–149     

Activity monitoring of functional OprM using a biomimetic microfluidic device

This paper describes the fabrication and use of a biomimetic microfluidic device for the monitoring of a functional porin reconstituted within a miniaturized suspended artificial bilayer lipid membrane (BLM). Such a microfluidic device allows for (1) fluidic and electrical access to both sides of the BLM and (2) reproducible membrane protein insertion and long-term electrical monitoring of its conductance (G(i)), thanks to the miniaturization of the BLM. We demonstrate here for the first time the feasibility to insert a large trans-membrane protein through its β-barrel, and monitor its functional activity for more than 1 hour (limited by buffer evaporation). In this paper, we specifically used our device for the monitoring of OprM, a bacterial efflux channel involved in the multidrug resistance of the bacteria Pseudomonas aeruginosa. Sub-steps of the OprM channel conductance were detected during the electrical recordings within our device, which might be due to oscillations between several structural conformations (sub-states) adopted by the protein, as part of its opening mechanism. This work is a first step towards the establishment of a genuine platform dedicated to the investigation of bacterial proteins under reconstituted conditions, a very promising tool for the screening of new inhibitors against bacterial channels involved in drug resistance.     

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.