Micro cell encapsulation and its hydrogel-beads production using microfluidic device

In this paper, we propose a cell encapsulation and hydrogel-beads production method using droplet formation in a microchannel. The hydrogel-beads produced by the microfluidic device developed here have smaller diameter and narrower distribution in their diameter compared to the conventional method, such as the droplet extrusion and the emulsification. The effects of the flow velocity and microchannel wall were analyzed based on fluid dynamical analysis. The results revealed that the wall effect of the microchannel strongly affected to the diameter of the droplet and the shape of the hydrogel-beads.     

Culture and chemical-induced fusion of tobacco mesophyll protoplasts in a microfluidic device

Microfluidic systems provide a powerful platform for biological analysis and have been applied in many disciplines. However, few efforts have been devoted to plant cell study. In this article, an optimized culture of tobacco mesophyll protoplasts and their first polyethylene glycol-induced fusion in a microfluidic device are presented. Culture medium optimization and dynamics of protoplast growth including size change, organelle motion, and cell mass formation were also investigated microscopically in real-time. On-chip protoplast culture showed that the first division percentage of tobacco mesophyll protoplasts could be improved as high as up to 85.6% in 5 days using NT1 medium, and the percentage of small cell mass formation was more than 48.0% in 10 days. Meanwhile, chemical-induced fusion of tobacco mesophyll protoplasts was realized in 3–5 min and a 28.8% fusion rate was obtained, which was similar to the conventional fusion in a macro-scale environment. These results will be helpful for the development of microfluidics-based studies on manipulation and analysis of plant cells in a miniaturized environment, including cell growth and differentiation, gene isolation, and cloning.     

Selective position of individual cells without lysis on a circular window array using dielectrophoresis in a microfluidic device

We trapped individual cells between two circular windows using negative dielectrophoretic (DEP) force and then sequentially trapped them inside circular windows by positive DEP force without electrical lysis in a microfluidic device. Three parameters, (1) the transmembrane potential that determines the lysis of a cell, (2) individual cell size that affects the trapped position accuracy of the cell, and (3) the Clausius–Mossotti (CM) factor that decides the trapped efficiency of the cell, were characterized experimentally and numerically in this sequential cell trapping technique. In this characterization, we confirmed that the swap rate of applied voltage frequency, size similarity between the cell and circular window, and instantaneous change rate of Re(f _CM) as a function of frequency were important factors in determining the selective position of individual cells without lysis. Our results provide useful suggestions for designing the structure of microfluidic DEP devices and optimizing variables required to manipulate individual cell trapping using both negative and positive DEP forces.     

3D-printed microfluidic manipulation device integrated with magnetic array

This paper reported a transparent, high-precision 3D-printed microfluidic device integrated with magnet array for magnetic manipulation. A reserved groove in the device can well constrain the Halbach array or conventional alternating array. Numerical simulations and experimental data indicate that the magnetic flux density ranges from 30 to 400 mT and its gradient is about 0.2–0.4 T/m in the manipulation channel. The magnetic field parameters of Halbach array in the same location are better than the other array. Diamagnetic polystyrene beads experience a repulsive force and move away from the magnetic field source under the effect of negative magnetophoresis. It is undeniable that as the flow rate increases, the ability of Halbach array to screen particle sizes decreases. Even so, it has a good particle size discrimination at a volumetric flow rate of 1.08 mL/h, which is much larger than that of a conventional PDMS device with a single magnet. The observed particle trajectories also confirm these statements. The deflection angle is related to the magnetic field, flow rate, and particle size. This 3D-printed device integrated with Halbach array offers excellent magnetic manipulation performance.     

Generation of disk-like hydrogel beads for cell encapsulation and manipulation using a droplet-based microfluidic device

We report a droplet-based microfluidic synthetic technique to generate disk-like hydrogel beads for cell encapsulation and manipulation. Utilizing this microfluidic synthetic technique, the size of the disk-like calcium alginate (CA) hydrogel beads and the number of cells encapsulated in the disk-like CA hydrogel beads could be well controlled by individually adjusting the flow rates of reagents. As a proof-of-concept, we demonstrated that single cell (yeast cell or mammalian cell) could be successfully encapsulated into disk-like CA hydrogel beads with high cell viability. Taking advantage of the flat top/bottom surfaces of disk-like CA hydrogel beads, cell division processes in culture media were clearly observed and recorded at a desired position without rolling and moving. This facile microfluidic chip provides a feasible method for size-controlled disk-like hydrogel beads generation and cell encapsulation. It could be a promising candidate for cell division observation and quantitative biological study in lab-on-a-chip applications.     

Continuous cell cross over and lysis in a microfluidic device

Complex sample preparation processes are major stumbling blocks for the development of lab-on-a-chip (LOC). We herein advance a microfluidic device for chemical cell lysis using a cell cross over (CCO) technology for the purpose of minimizing the sample preparation steps. The proposed device allows cells to continuously cross over from a cell carrier to a cell lysis solution in a CCO region and to be automatically lysed. For the successful CCO and cell lysis, microflow patterns and cell movements in the CCO region are investigated by experimental as well as numerical studies. EL-4 mammalian cells are used for the demonstration of the performance of the proposed device. The DNA sample obtained from the developed device is quantitatively and qualitatively compared with the one obtained from a conventional chemical cell lysis method by using a UV–Vis spectrophotometer and gel electrophoresis. The quantitative analysis shows that the recovered DNA is 86% compared to the one obtained from the conventional chemical cell lysis.     

A microfluidic device for array patterning by perpendicular electrokinetic focusing

This paper describes a microfluidic chip in which two perpendicular laminar-flow streams can be operated to sequentially address the surface of a flow-chamber with semi-parallel sample streams. The sample streams can be controlled in position and width by the method of electrokinetic focusing. For this purpose, each of the two streams is sandwiched by two parallel sheath flow streams containing just a buffer solution. The streams are being electroosmotically pumped, allowing a simple chip design and a setup with no moving parts. Positioning of the streams was adjusted in real-time by controlling the applied voltages according to an analytical model. The perpendicular focusing gives rise to overlapping regions, which, by combinatorial (bio) chemistry, might be used for fabrication of spot arrays of immobilized proteins and other biomolecules. Since the patterning procedure is done in a closed, liquid filled flow-structure, array spots will never be exposed to air and are prevented from drying. With this device configuration, it was possible to visualize an array of 49 spots on a surface area of 1 mm². This article describes the principle, fabrication, experimental results, analytical modeling and numerical simulations of the microfluidic chip.     

Quantitative analysis of sperm rheotaxis using a microfluidic device

Quite puzzling issue in biology is how sperm cells are selected naturally where human sperm has to maintain a correct swimming behavior during the various stages of reproduction process. In nature, sperm has to compete a long journey from cervix to oocyte to stand a chance for fertilization. Although various guidance mechanisms such as chemical and thermal gradients are proposed previously, these mechanisms may only be relevant as sperm reaches very close to the oocyte. Rheotaxis, a phenomenon where sperm cells swim against the flow direction, is possibly the long-range sperm guidance mechanism for successful fertilization. A little is known quantitatively about how flow shear effects may help guide human sperm cells over long distances. Here, we have developed microfluidic devices to quantitatively investigate sperm rheotaxis at various physiological flow conditions. We observed that at certain flow rates sperm actively orient and swim against the flow. Sperm that exhibit positive rheotaxis show better motility and velocity than the control (no-flow condition), however, rheotaxis does not select sperm based on hyaluronic acid (HA) binding potential and morphology. Morphology and HA binding potential may not be a significant factor in sperm transport in natural sperm selection.     

Design and fabrication of a microfluidic device for near-single cell mRNA isolation using a copper hot embossing master

We describe investigations toward a disposable polymer-based chip for the isolation of eukaryotic mRNA. This work focuses here on the improvement of the fabrication methods for rapid prototyping and the actual application at lowest RNA concentrations with total channel volumes of 3.5 μL. Messenger RNA isolation was achieved using paramagnetic oligo (dT)₂₅ beads within a microfluidic channel which incorporated a sawtooth microstructured design to aid in mixing. The structures were shown to facilitate mixing beteen two fluids in parallel flow when compared to a channel without structures. The chip was fabricated by means of hot embossing poly(methyl methacrylate) (PMMA) using a copper master. Copper was used as the master material due to its excellent thermal, mechanical, and electroplating properties. Fabrication of the master consisted of the structuring of a polished copper plate using KMPR 1050 as an electroplating mold for forming the microchannel structures. The copper master was found to be much more robust than traditional silicon masters used for prototyping. The use of KMPR enabled the generation of high straight walls in contrast to SU-8 masters. In addition, embossing times were able to be decreased by a factor of 3 due to improved heat conduction and avoidance of a lengthy and delicate de-embossing step.     

Enhanced-throughput production of polymersomes using a parallelized capillary microfluidic device

We report a parallelized capillary microfluidic device for enhanced production rate of monodisperse polymersomes. This device consists of four independent capillary microfluidic devices, operated in parallel; each device produces monodisperse water-in-oil-in-water (W/O/W) double-emulsion drops through a single-step emulsification. During generation of the double-emulsion drops, the innermost water drop is formed first and it triggers a breakup of the middle oil phase over wide range of flow rates; this enables robust and stable formation of the double-emulsion drops in all drop makers of the parallelized device. Double-emulsion drops are transformed to polymersomes through a dewetting of the amphiphile-laden middle oil phase on the surface of the innermost water drop, followed by the subsequent separation of the oil drop. Therefore, we can make polymersomes with a production rate enhanced by a factor given by the number of drop makers in the parallelized device.     

High throughput blood plasma separation using a passive PMMA microfluidic device

Since plasma is rich in many biomarkers used in clinical diagnostic experiments, microscale blood plasma separation is a primitive step in most of microfluidic analytical chips. In this paper, a passive microfluidic device for on-chip blood plasma separation based on Zweifach–Fung effect and plasma skimming was designed and fabricated by hot embossing of microchannels on a PMMA substrate and thermal bonding process. Human blood was diluted in various times and injected into the device. The main novelty of the proposed microfluidic device is the design of diffuser-shaped daughter channels. Our results demonstrated that this design exerted a considerable positive influence on the separation efficiency of the passive separator device, and the separation efficiency of 66.6 % was achieved. The optimum purity efficiency of 70 % was achieved for 1:100 dilution times.     

A cell electrofusion microfluidic chip with micro-cavity microelectrode array

A new cell electrofusion microfluidic chip with 19,000 pairs of micro-cavity structures patterned on vertical sidewalls of a serpentine-shaped microchannel has been designed and fabricated. In each micro-cavity structure, the two sidewalls perpendicular to the microchannel are made of SiO₂ insulator, and that parallel to the microchannel is made of silicon as the microelectrode. One purpose of the design with micro-cavity microelectrode array is to obtain high membrane voltage occurring at the contact point of two paired cells, where cell fusion takes place. The device was tested to electrofuse NIH3T3 and myoblast cells under a relatively low voltage (~9 V). Under an AC electric field applied between the pair of microelectrodes positioned in the opposite micro-cavities, about 85–90 % micro-cavities captured cells, and about 60 % micro-cavities are effectively capable of trapping the desired two-cell pairs. DC electric pulses of low voltage (~9 V) were subsequently applied between the micro-cavity microelectrode arrays to induce electrofusion. Due to the concentration of the local electric field near the micro-cavity structure, fusion efficiency reaches about 50 % of total cells loaded into the device. Multi-cell electrofusion and membrane rupture at the end of cell chains are eliminated through the present novel design.     

Sheathless microfluidic particle focusing technique using slanted microstructure array

This paper proposes a microfluidic channel for particle focusing that uses a microstructure on the bottom of the channel. Particles can be effectively focused in channels with bottom structures because of microvortex induced by the structure. Microchannels with top structures (top type) and bottom structures (bottom type) were fabricated. The focusing ratios in the focusing region (one-eighth of the channel width) were 86 % in top type and 89 % in bottom type at a flow rate of 1 μl/min. When the flow rate was increased to 5 μl/min, particles in top type were barely focused, whereas particles in bottom type were focused with a focusing ratio of approximately 80 %. We also evaluated the effect of a slanted angle for the microstructures. The comparative experiment was conducted with microstructures fabricated at slanted angle intervals of 20° (20°, 40°, 60°, and 80°) and 10°. The results indicated that the slanted angle (20°) required a small number of microstructures to direct the sample to the focusing region. For microstructures with a 20° slanted angle, the sample was focused after passing through 20 microstructures (10 mm). However, microstructures with an angle of 80° needed over 70 microstructures (over 23 mm) to direct the particle. In this sense, a microchannel with microstructures slanted at 20° is applicable to miniaturized devices. These results show that the microchannel with bottom structures slanted 20° can be used to effectively focus samples with advantages of applying various ranges of flow rates and miniaturizing devices.     

DNA ligation using a disposable microfluidic device combined with a micromixer and microchannel reactor

A novel PDMS and glass-based microfluidic device consisting of a micromixer and microreactor for DNA ligation is described in this article. The new passive type planar micromixer is 10.33 mm long and composed of a straight channel integrated with nozzles and pillars, and the microreactor is composed of a serpentine channel. Mixing was enhanced by convective diffusion facilitated by the nozzles and pillars. The performance of the micromixer was analytically simulated and experimentally evaluated. The micromixer showed a good mixing efficiency of 87.7% at a 500 μL/min flow rate (Re = 66.5). DNA ligation was successfully performed using the new microfluidic device, and ligation time was shortened from 4 h to 5 min. When used for on-chip ligation, this new micromixer offers advantages of disposability and portability.     

A microfluidic device for chemical and mechanical stimulation of mesenchymal stem cells

Recently, there has been considerable interest in stem cells which have the potential to differentiate into multiple lineages for cell therapy. Special attention has been paid, in particular, to the use of alternative sources of stem cells with fewer ethical issues. For example, mesenchymal stem cells (MSCs) from bone marrow have been proven to be multipotent for transplantation and tissue engineering. In this study, an integrated microfluidic chip capable of chemically and mechanically stimulating human mesenchymal stem cells (hMSCs) for adipogenic differentiation was presented. It was composed of a dilution module for controlling the insulin concentrations, and pneumatically-modulated membrane structures for precisely applying shear stresses on cells to perform chemical and mechanical stimulation, respectively. With this approach, a long-term culture and differentiation of MSCs were performed in an automatic manner. The accumulation of lipids that represented the results of insulin simulation was evaluated by Oil Red O staining. The measurements including lipid droplet numbers, optical density (OD) values, and expression of the PPARγ2 gene were used to assess the level of differentiation of the MSCs. The experimental result showed that the maximum oil droplets were induced under an optimal insulin concentration of 10 μg/ml. It was also revealed that, under mechanical stimulation, adipogenesis was inhibited under stronger levels of applied shear stresses and at higher pulsation frequencies. This proposed microfluidic chip has great potential as a powerful tool for MSC studies.     

Rapid measurement of fluid viscosity using co-flowing in a co-axial microfluidic device

This article presents a simple microfluidic method to measure the Newtonian fluid viscosity. This method is carried out in a co-axial microfluidic device. A stable liquid/liquid annular co-laminar flow in the co-axial microfluidic device has been realized, which can be described by Navier–Stokes equations. The viscosity of either fluid can be measured based on the equations when the viscosities of another fluid is known. Proper conditions to form stable annular co-laminar flow for the viscosity measurement were investigated. Several fluids were tested with viscosity ranging from 0.6 to 40 mPa s. The measured results fit very well with those measured by a commercial spinning digital viscometer. The novel method is highly controllable and reliable, and has the advantage of less time and material consumption, as well as easy fabrication of the device.     

Fabrication of microfluidic device channel using a photopolymer for colloidal particle separation

We have developed a method of fabricating microfluidic device channels for bio-nanoelectronics system by using high performance epoxy based dry photopolymer films or dry film resists (DFRs). The DFR used was with a trademark name Ordyl SY355 from Elga Europe. The developing and exposing processes as well as the time taken in making the channels are recorded. Finally from those recorded methods, the accurate procedures and time taken for DFR development and exposure have been found and ultimately been consistently used in fabricating our channels. These channels were patterned and sandwiched in between two glass substrates. In our advance, the channel was formed for the colloidal particle separation system. They can be used for handling continuous fluid flow and particle repositioning maneuver using dielectrophoresis that have showed successful results in the separation.     

Automating fluid delivery in a capillary microfluidic device using low-voltage electrowetting valves

A multilayer capillary polymeric microfluidic device integrated with three normally closed electrowetting valves for timed fluidic delivery was developed. The microfluidic channel consisted two flexible layers of poly (ethylene terephthalate) bonded by a pressure-sensitive adhesive spacer tape. Channels were patterned in the spacer tape using laser ablation. Each valve contained two inkjet-printed silver electrodes in series. Capillary flow within the microchannel was stopped at the second electrode which was modified with a hydrophobic monolayer (valve closed). When a potential was applied across the electrodes, the hydrophobic monolayer became hydrophilic and allowed flow to continue (valve opened). The relationship between the actuation voltage, the actuation time, and the distance between two electrodes was performed using a microfluidic chip containing a single microchannel design. The results showed that a low voltage (4.5 V) was able to open the valve within 1 s when the distance between two electrodes was 1 mm. Increased voltages were needed to open the valves when the distance between two electrodes was increased. Additionally, the actuation time required to open the valve increased when voltage was decreased. A multichannel device was fabricated to demonstrate timed fluid delivery between three solutions. Our electrowetting valve system was fabricated using low-cost materials and techniques, can be actuated by a battery, and can be integrated into portable microfluidic devices suitable for point-of-care analysis in resource-limited settings.