Establishment of a confluent cardiomyocyte culture in a cylindrical microchannel

We established a confluent cardiomyocyte culture method using an 800-μm diameter cylindrical microchannel in this report. This was realized by introducing cardiomyocytes 2 times before and after turning over a microchip. The optimum condition was starting the flowing medium 2.0 h after seeding and flowing the medium at 1.0 μL/min. By applying this technology to a cardiomyocyte-based spherical heart pump device, one may develop self-fluid regulated devices that could be applied for implantable or circulation analysis device on a chip.     

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.     

Understanding microchannel culture: parameters involved in soluble factor signaling

While the importance of autocrine-paracrine signaling in vivo is clear, the ability to study the effects of secreted endogenous factors in vitro is hampered by canonical culture platforms. In multi-well plates, the large air-liquid interface gives rise to convective flows that continually mix the fluid disrupting the local diffusion-based accumulation. Simple microchannels provide a more controlled microenvironment that can be used to study secreted factor effects. Here, we utilize microchannel culture to examine basic culture parameters and their interactions using normal mammary gland epithelial cells (NMuMG). The following parameters were studied: (1) cell density (80 vs. 240 cells mm(-2)), (2) exogenous growth factors (epidermal growth factor [EGF] vs. fetal bovine serum), (3) medium change frequency (1 h, 4 h, 12 h), and (4) culture platform (microchannels vs. 96-well plates). The cells exhibited increased growth rates in microchannels as compared to 96-well plates. Cell proliferation increased as the frequency of media change decreased. For the microchannel geometries used, important threshold concentrations were reached in a few hours. In aggregate, the results indicate that the function of the four factors and their interactions on NMuMG growth are spatially and temporally related by molecular diffusion in the controlled microchannel space. The convective-free microchannel environment may prove useful for studying soluble factor signaling in vitro, and to test models and predictions of autocrine-paracrine signaling.     

Embryonic development in the mouse is enhanced via microchannel culture

Microfluidic devices (microchannels) have been fabricated and tested for embryo culture. Three different microfabrication materials (silicon, polydimethylsiloxane (PDMS), and borosilicate) were used to fabricate the microchannels. The objective of this study was to determine if static microchannels permitted culture of mouse embryos to the blastocyst stage. Groups of 10 two-cell ICR x B6SJL/F1 mouse embryos were cultured for 96 hours in 4 different physical culture systems: 1) silicon/borosilicate microchannels, 2) PDMS/borosilicate microchannels, and 3) standard microdrops. Embryos cultured in the silicon/borosilicate and PDMS/borosilicate microchannels exhibited a faster rate of cleavage (P < 0.05), and produced more blastocysts (P < 0.01) than control microdrops. Furthermore, microchannels had a lower percentage of degenerated embryos than control embryos (P < 0.01). The results suggest that the microchannel culture systems may provide a culture environment that more closely mimics the in vivo environment.     

A method for DNA detection in a microchannel: fluid dynamics phenomena and optimization of microchannel structure

A simple DNA diagnosis method using microfluidics has been developed which requires simple and straightforward procedures such as injection of sample and probe DNA solutions. This method takes advantage of the highly accurate control of fluids in microchannels, and is superior to DNA microarray diagnosis methods due to its simplicity, highly quantitative determination, and high-sensitivity. The method is capable of detecting DNA hybridization for molecules as small as a 20 mer. This suggests the difference in microfluidic behavior between single strand DNA (ssDNA) and double stranded DNA (dsDNA). In this work, influence of both the inertial force exerted on DNA molecules and the diffusion of DNA molecules was investigated. Based on the determination of these parameters for both ssDNA and dsDNA by experiments, a numerical model describing the phenomena in the microchannel was designed. Computational simulation results using this model were in good agreement with previously reported experimental results. The simulation results showed that appropriate selection of the analysis point and the design of microchannel structure are important to bring out the diffusion and inertial force effects suitably and increase the sensitivity of the detection of DNA hybridization, that is, the analytical performance of the microfluidic DNA chip.     

Normal human dermal fibroblasts: proteomic analysis of cell layer and culture medium.

Proteins present within the cell layer and those released in the cell medium from in vitro cultured normal human dermal fibroblasts were separated and characterized in terms of their isoelectric point and molecular weight, by two-dimensional (2-D) gel electrophoresis. All spots in the synthetic gel were firstly analyzed by the Melanie 3 software and compared with those of breast cancer cells, colorectal epithelial cells, HL60, lymphoma cells, and platelets, already available on-line. From the identification of 144 spots from both the cell layer and the medium, we were able to recognize 89 different proteins, since a certain number of spots represented different isoforms of the same molecule. Identifications were performed by matching with on-line 2-D databases, and by matrix assisted laser-desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), in order to confirm the identification by matching, or to identify new proteins. The procedure we used allows (i) to design a highly reproducible reference map of the proteome of adult human normal fibroblasts in culture, (ii) to evaluate protein species produced in the cell layer as well as those released in the culture medium, and (iii) to compare data from gel matching with those obtained by MS. This work represents an essential step for a better knowledge of mesenchymal cells, given the widespread use of this cell type in both clinical and experimental investigations.     

Droplet formation in a microchannel network

A method is given for generating droplets in a microchannel network. With oil as the continuous phase and water as the dispersed phase, pico/nanoliter-sized water droplets can be generated in a continuous phase flow at a -junction. The channel for the dispersed phase is 100 microm wide and 100 microm deep, whereas the channel for the continuous phase is 500 microm wide and 100 microm deep. For given experimental parameters, regular-sized droplets are reproducibly formed at a uniform speed. The diameter of these droplets is controllable in the range from 100-380 microm as the flow velocity of the continuous phase is varied from 0.01 m s(-1) to 0.15 m s(-1).     

Dynamics of fibers in a wide microchannel

Dynamics of single flexible non-Brownian fibers, tumbling in a Poiseuille flow between two parallel solid plane walls, is studied with the use of the HYDROMULTIPOLE numerical code, based on the multipole expansion of the Stokes equations, corrected for lubrication. Fibers, which are closer to a wall, more flexible (less stiff) or longer, deform more significantly and, for a wide range of the system parameters, they faster migrate towards the middle plane of the channel. For the considered systems, fiber velocity along the flow is only slightly smaller than (and can be well approximated by) the Poseuille flow velocity at the same position. In this way, the history of a fiber migration across the channel is sufficient to determine with a high accuracy its displacement along the flow.     

Heat transfer intensification in microchannel by induced-charge electrokinetic phenomenon: a numerical study

Journal of Thermal Analysis and Calorimetry - In this numerical investigation, the induced-charge electrokinetic phenomenon is used to intensify the convective heat transfer rate in the...     

Brownian dynamics simulation and experimental study of colloidal particle deposition in a microchannel flow

This paper reports an analysis of the irreversible deposition of colloidal particles from the pressure-driven flow in a microchannel within the framework of DLVO theory. A theoretical model is presented on the basis of the stochastic Langevin equation, incorporating the random Brownian motion of colloidal particles. Brownian dynamics simulation is used to compute the particle deposition in terms of the surface coverage. To validate the theoretical model, experiments are carried out using the parallel-plate flow cell technique, enabling direct videomicroscopic observation of the deposition kinetics of polystyrene latex particles in NaCl electrolytes. The theoretical predictions are compared with the experimental results, and good agreement is found.     

Dipolophoresis of Janus nanoparticles in a microchannel

A nonlinear dipolophoretic analysis is applied to analytically explain the counterintuitive experimental results of Gangwal et al. [16, 17] that an uncharged micro/nanosize dielectric Janus particle is attracted to the wall of a microchannel when exposed to an AC‐uniform electric field in the direction parallel to the no‐slip boundaries. We employ the so‐called “weak” field assumption and consider a metallodielectric Janus colloid comprising two semispheres of distinct dielectric properties subject to an oscillating‐uniform electric field with moderate frequency (below the Maxwell–Wagner limit). The Debye scale (ratio of electric double layer thickness to particle size) is considered unrestricted. Under the low Reynolds number hypothesis, Faxén's theorem and the Green's function (Stokeslet) method of singularities, including appropriate images with respect to the no‐slip boundary, are applied under the remote‐field approximation to determine the dynamics and trajectory of a small colloid moving near a wall. When assuming maximum dielectric contrasts between hemispheres and relatively low Debye scale (compared to particle radius), a rather simple relation for the equilibrium position of the colloid (i.e. tilt angle and distance from the wall) is obtained and found to be in qualitative good agreement with the experimental observations of Gangwal et al. [16, 17] and the predictions of Kilic and Bazant [12].     

DNA separation by EFFF in a microchannel

This paper theoretically explores the application of electric field flow fractionation (EFFF) for the size-based separation of DNA strands in a microchannel. An axial electric field cannot separate DNA strands in solution because the electrical mobility of the strands is independent of the length. However, lateral electric fields coupled with an axial Poiseuille flow can separate the DNA strands of different sizes. By using regular perturbation analysis, we obtain the effective diffusivity and the mean velocity of the DNA molecules that are undergoing a pressure driven Poiseuille flow in a 2D channel in presence of a lateral electric field. The mean velocities and the dispersion coefficients are then utilized to determine the scaling for length of the channel and the time required for separation of DNA molecules in different parameter regimes. The results show that EFFF can separate DNA strands in the range of 10 kbp that differ in size by about 2.5 kbp in about half an hour in a 1 cm long channel. While DNA strands can be separated by EFFF, the performance of devices based on EFFF seems to be at best comparable to other techniques such as entropic trapping.     

Identification of a phosphoprotein that is downregulated in immortalized human fibroblasts

Many lines of evidence indicate that the immortalization step is critical for the neoplastic transformation of normal human cells. Once normal human cells have been immortalized, they are relatively easily transformed into neoplastic cells. In order to understand these phenomena, patterns of protein phosphorylation in proliferating normal human fibroblast cell strains and their immortalized cell lines were compared by using two-dimensional polyacrylamide gel electrophoresis. It was found that the expression and phosphorylation levels of the human heat shock protein 27 (HSP27) were predominantly downregulated in the immortalized cells compared with those in their normal counterparts. In the normal cells, HSP27 expression and phosphorylation were markedly increased by physiological and nonphysiological stresses, such as serum addition, treatment with a carcinogenic agent like 4-nitroquinoline-1-oxide, and a high osmotic pressure. This may be a normal defense against acute changes of cellular environment and cytotoxic effects. However, these stresses had no effects on the expression and phosphorylation of HSP27 in the immortalized cells. These results suggest that an abnormal regulation of HSP27 expression and phosphorylation may be one of the reasons for easy neoplastic transformation of the immortalized cells by the treatment with carcinogenic agents.     

Inertial separation in a contraction-expansion array microchannel

We report a contraction-expansion array (CEA) microchannel that allows inertial size separation by a force balance between inertial lift and Dean drag forces in fluid regimes in which inertial fluid effects become significant. An abrupt change of the cross-sectional area of the channel curves fluid streams and produces a similar effect compared to Dean flows in a curved microchannel of constant cross-section, thereby inducing Dean drag forces acting on particles. In addition, the particles are influenced by inertial lift forces throughout the contraction regions. These two forces act in opposite directions each other throughout the CEA microchannel, and their force balancing determines whether the particles cross the channel, following Dean flows. Here we describe the physics and design of the CEA microfluidic device, and demonstrate complete separation of microparticles (polystyrene beads of 4 and 10 μm in diameter) and efficient exchange of the carrier medium while retaining 10 μm beads.     

A new droplet breakup phenomenon in electrokinetic flow through a microchannel constriction

A completely new droplet breakup phenomenon is reported for droplets passing through a constriction in an electrokinetic flow. The breakup occurs during the droplet shape recovery process past the constriction throat by the interplay of the dielectrophoretic stress release and the interface energy for droplets with smaller permittivity than that of the ambient fluid. There are conditions for constriction ratios and droplet size that the droplet breakup occurs. The numerical predictions provided here require experimental verification, and then can give rise to a novel microfluidic device design with novel droplet manipulations.     

DNA sequencing in a monolithic microchannel device

We present 50 cm long microchannels in a monolithic device for high resolution, long read-length DNA sequencing. These devices were fabricated and bonded in borofloat glass using unconventional photolithography techniques with 48-188 independent, straight microchannels. The microchannel DNA separation was tested with POP-6 polymer and a DNA sequencing ladder separated at room temperature and 200 V/cm. Single-base resolution greater than 600 bases was achieved and the sequence base called to 640 bases with 98% accuracy. Under the same experimental conditions, the performance of the microchip was identical to a fused-silica capillary with similar cross-sectional area.     

A miniature gas analyzer made by integrating a chemoresistor with a microchannel

A microfluidic channel is integrated with a tin oxide-based generic gas sensor on a PMMA (polymethyl methacrylate) substrate to fabricate a miniature gas analyzer. The analyte gas diffuses along the air-filled channel to affect the sensor installed in a microcavity positioned at the end of the channel. Analyte diffusion rates, experimentally estimated based on the temporal responses received from the sensor, are connected to the analyte's interactions with the channel walls as well as its diffusivity in air. The analyte-related information is extracted from the recorded responses and used for analyte recognition. A single PMMA channel of 80 μm × 3 mm × 50 mm dimensions facilitates the correct classification of single component contaminants each introduced in a wide concentration range in air. The device is also shown to identify 15 ppm of 2-butanol in air contaminated with 1500 ppm of 1-butanol. The gas analyzer fabricated based on this concept is durable, inexpensive, handheld and suitable for a variety of applications.     

Experimental and theoretical study of the displacement process between two electrolyte solutions in a microchannel

Displacement of one electrolyte solution by another in a microchannel is required in many biolab chip devices. The objective of this paper is to develop a better understanding of the displacement process between two electrolyte solutions under an applied electric field in a cylindrical microchannel in terms of the traveling distance of the interface between these two electrolyte solutions. In order to develop a general model to predict the location of the interface, two different situations are considered; one model assumes the presence of a sharp interface between the two solutions and the other model considers a mixing zone between the two solutions. Carefully conducted experiments were carried out to obtain the current-time relationship, which is used in the model to predict the location of the interface. In these experiments, deionized ultrafiltered water (DIUF water), 10 mM KCl, 0.1 mM KCl, and 0.1 mM LaCl3 solutions were used as the testing liquids. Polyamide-coated silica capillary tubes of internal diameter 100 mum and length 10 cm were employed in this study. The relationship between traveled distance of the interface and time was predicted by a developed model based on the measured current-time relationship for such a displacement process under a constant applied electric field. The characteristics of the nonlinear change of the traveling distance with the time were also discussed in this paper.