Identification of Single Yeast Budding Using Impedance Cytometry with a Narrow Electrode Span.

Impedance cytometry is wildly used in single-cell detection, and its sensitivity is essential for determining the status of single cells. In this work, we focus on the effect of electrode gap on detection sensitivity. Through comparing the electrode span of 1 µm and 5 µm, our work shows that narrowing the electrode span could greatly improve detection sensitivity. The mechanism underlying the sensitivity improvement was analyzed via numerical simulation. The small electrode gap (1 µm) allows the electric field to concentrate near the detection area, resulting in a high sensitivity for tiny particles. This finding is also verified with the mixture suspension of 1 µm and 3 µm polystyrene beads. As a result, the electrodes with 1 µm gap can detect more 1 µm beads in the suspension than electrodes with 5 µm gap. Additionally, for single yeast cells analysis, it is found that impedance cytometry with 1 µm electrodes gap can easily distinguish budding yeast cells, which cannot be realized by the impedance cytometry with 5 µm electrodes gap. All experimental results support that narrowing the electrode gap is necessary for tiny particle detection, which is an important step in the development of submicron and nanoscale impedance cytometry.

Sheathless Inertial Focusing Chip Combining a Spiral Channel with Periodic Expansion Structures for Efficient and Stable Particle Sorting.

Efficient and reliable manipulation of biological particles is crucial in medical diagnosis and chemical synthesis. Inertial microfluidic devices utilizing passive hydrodynamic forces in the secondary flow have drawn considerable attention for their high throughputs, low costs, and harmless particle manipulation. However, as the dominant mechanism, the inertial lift force is difficult to quantitatively analyze because of the uncertainties of its magnitude and direction. The equilibrium position of particles varies along the migration process, thus inducing the instabilities of particle separation. Herein, we present a designable inertial microfluidic chip combining a spiral channel with periodic expansion structures for the sheathless separation of particles with different sizes. The stable vortex-induced lift force arising from the periodic expansion and the Dean drag force significantly enhanced the focusing process and determined the final equilibrium position. The experimental results showed that over 99% of target particles could be isolated with the high target sample purity of 86.12%. In the biological experiment, 93.5% of the MCF-7, 89.5% of the Hela, and 88.6% of the A549 cells were steadily recovered with excellent viabilities to verify the potential of the device in dealing with biological particles over a broad range of throughputs. The device presented in this study can further serve as a lab-on-chip platform for liquid biopsy and diagnostic analysis.

User-friendly cell patterning methods using a polydimethylsiloxane mold with microchannels.

Techniques for partitioning cell adhesion are useful tools in biological and medical experiments. However, conventional cell patterning methods require special apparatus, special materials or high-level skills. Therefore, we have developed a new cell patterning methodology which can be easily carried out in biological laboratories. Non-cell adhesive material including hydrogel or gas patterns to restrict cell adhesion on a culture dish or glass substrates can be constructed by exploiting a polydimethylsiloxane (PDMS) mold with microchannels. The PDMS molds suck non-adhesive materials into microchannels from the inlet of the microchannels and the materials are immobilized onto the substrates with a desired pattern. High resolution under a few micrometers and long-term stability can be realized. This method has been used for analysis of stem cells, muscle cells, neuron development and other cells in collaboration with many biological researchers. Several examples to use this technique are introduced in this review.

Dual-frequency impedance assays for intracellular components in microalgal cells.

Intracellular components (including organelles and biomolecules) at the submicron level are typically analyzed in situ by special preparation or expensive setups. Here, a label-free and cost-effective approach of screening microalgal single-cells at a subcellular resolution is available based on impedance cytometry. To the best of our knowledge, it is the first time that the relationships between impedance signals and submicron intracellular organelles and biomolecules are shown. Experiments were performed on Euglena gracilis (E. gracilis) cells incubated under different incubation conditions (i.e., aerobic and anaerobic) and 15 µm polystyrene beads (reference) at two distinct stimulation frequencies (i.e., 500 kHz and 6 MHz). Based on the impedance detection of tens of thousands of samples at a throughput of about 900 cells per second, three metrics were used to track the changes in biophysical properties of samples. As a result, the electrical diameters of cells showed a clear shrinkage in cell volume and intracellular components, as observed under a microscope. The morphology metric of impedance pulses (i.e., tilt index) successfully characterized the changes in cell shape and intracellular composition distribution. Besides, the electrical opacity showed a stable ratio of the intracellular components to cell volume under the cellular self-regulation. Additionally, simulations were used to support these findings and to elucidate how submicron intracellular components and cell morphology affect impedance signals, providing a basis for future improvements. This work opens up a label-free and high-throughput way to analyze single-cell intracellular components by impedance cytometry.

Shape-based separation of drug-treated Escherichia coli using viscoelastic microfluidics.

Here, we achieve shape-based separation of drug-treated Escherichia coli (E. coli) by viscoelastic microfluidics. Since shape is critical for modulating biological functions of E. coli, the ability to prepare homogeneous E. coli populations adopting uniform shape or sort bacterial sub-population based on their shape has significant implications for a broad range of biological, biomedical and environmental applications. A proportion of E. coli treated with 1 µg mL-1 of the antibiotic mecillinam were found to exhibit changes in shape from rod to sphere, and the heterogeneous E. coli populations after drug treatment with various aspect ratios (ARs) ranging from 1.0 to 5.5 were used for experiment. We demonstrate that E. coli with a lower AR, i.e., spherical E. coli (AR ≤ 1.5), are directed toward the middle outlet, while rod-shaped E. coli with a higher AR (AR > 1.5) are driven to the side outlets. Further, we demonstrate that the separation performance of the viscoelastic microfluidic device is influenced by two main factors: sheath-to-sample flow rate ratio and the concentration of poly-ethylene-oxide (PEO). To the best of our knowledge, this is the first report on shape-based separation of a single species of cells smaller than 4 µm by microfluidics.

Parallel Impedance Cytometry for Real-Time Screening of Bacterial Single Cells from Nano- to Microscale.

The benefits of impedance cytometry include high-throughput and label-free detection, while long-term calibration is required to remove the effects of the detection circuits. This study presents a novel impedance cytometry system, called parallel impedance cytometry, to simplify the calibration and analysis of the impedance signals. Furthermore, target objects can be detected even when benchmarked against similar objects. Parallel dual microchannels allow the simultaneous detection of reference and target particles in two separate microchannels, without the premixing of reference and target suspensions. The impedance pulses of both can appear separately on the opposite sides of the same time series, which have been verified via simulation and experimental results. Raw impedance signals can easily distinguish target particles from reference ones. Polystyrene beads with different sizes ranging from nano- to microscale (e.g., 500, 750 nm, 1, 2, 3, and 4.5 µm) confirm the nanosensitivity of the system. In addition, the detection of antibiotic-treated Escherichia coli cells demonstrates that our system can be used for the quantitative assessment of the dielectric properties of individual cells, as well as for the proportion of susceptible cells. Through benchmarking against untreated E. coli cells in the other channel, our method enables the discrimination of susceptible cells from others and the comparison of susceptible and insusceptible cells in the target suspension. Those findings indicate that the parallel impedance cytometry can greatly facilitate the measurement and calibration of the impedances of various particles or cells and provide a means to compare their dielectric properties.

A simple and reversible glass-glass bonding method to construct a microfluidic device and its application for cell recovery.

Compared with polymer microfluidic devices, glass microfluidic devices have advantages for diverse lab-on-a-chip applications due to their rigidity, optical transparency, thermal stability, and chemical/biological inertness. However, the bonding process to construct glass microfluidic devices usually involves treatment(s) like high temperature over 400 °C, oxygen plasma or piranha solution. Such processes require special skill, apparatus or harsh chemicals, and destroy molecules or cells in microchannels. Here, we present a simple method for glass-glass bonding to easily form microchannels. This method consists of two steps: placing water droplets on a glass substrate cleaned by neutral detergent, followed by fixing a cover glass plate on the glass substrate by binding clips for a few hours at room temperature. Surface analyses showed that the glass surface cleaned by neutral detergent had a higher ratio of SiOH over SiO than glass surfaces prepared by other cleaning steps. Thus, the suggested method could achieve stronger glass-glass bonding via dehydration condensation due to the higher density of SiOH. The pressure endurance reached over 600 kPa within 6 h of bonding, which is sufficient for practical microfluidic applications. Moreover, by exploiting the reversibility of this bonding method, cell recoveries after cultivating cells in a microchannel were demonstrated. This new bonding method can significantly improve both the productivity and the usability of glass microfluidic devices and extend the possibility of glass microfluidic applications in future.

Microscopic impedance cytometry for quantifying single cell shape.

In this work, we investigated the ability of impedance flow cytometry to measure the shape of single cells/particles. We found that the impedance pulses triggered by micro-objects that are asymmetric in morphology show a tilting trend, and there is no such a tilting trend for symmetric ones. Therefore, we proposed a new metric, tilt index, to quantify the tilt level of the impedance pulses. Through simulation, we found that the value of tilt index tends to be zero for perfectly symmetrical objects, while the value is greater than zero for asymmetrical ones. Also, this metric was found to be independent on the trajectories (i.e., lateral, and z-direction shift) of the target micro-object. In experiments, we adopted a home-made lock-in amplifier and performed experiments on 10 µm polystyrene beads and Euglena gracilis (E. gracilis) cells with varying shapes. The experimental results coincided with the simulation results and demonstrated that the new metric (tilt index) enables the impedance cytometry to characterize the shape single cells/particles without microscopy or other optical setups.

Focusing of Particles in a Microchannel with Laser Engraved Groove Arrays.

Continuous microfluidic focusing of particles, both synthetic and biological, is significant for a wide range of applications in industry, biology and biomedicine. In this study, we demonstrate the focusing of particles in a microchannel embedded with glass grooves engraved by femtosecond pulse (fs) laser. Results showed that the laser-engraved microstructures were capable of directing polystyrene particles and mouse myoblast cells (C2C12) towards the center of the microchannel at low Reynolds numbers (Re < 1). Numerical simulation revealed that localized side-to-center secondary flows induced by grooves at the channel bottom play an essential role in particle lateral displacement. Additionally, the focusing performance proved to be dependent on the angle of grooves and the middle open space between the grooves based on both experiments and simulation. Particle sedimentation rate was found to critically influence the focusing of particles of different sizes. Taking advantage of the size-dependent particle lateral displacement, selective focusing of micrometer particles was demonstrated. This study systematically investigated continuous particle focusing in a groove-embedded microchannel. We expect that this device will be used for further applications, such as cell sensing and nanoparticle separation in biological and biomedical areas.

Flow analysis on microcasting with degassed polydimethylsiloxane micro-channels for cell patterning with cross-linked albumin.

Patterned cell culturing is one of the most useful techniques for understanding the interaction between geometric conditions surrounding cells and their behaviors. The authors previously proposed a simple method for cell patterning with an agarose gel microstructure fabricated by microcasting with a degassed polydimethylsiloxane (PDMS) mold. Although the vacuum pressure produced from the degassed PDMS can drive a highly viscous agarose solution, the influence of solution viscosity on the casting process is unknown. This study investigated the influences of micro-channel dimensions or solution viscosity on the flow of the solution in a micro-channel of a PDMS mold by both experiments and numerical simulation. It was found experimentally that the degassed PDMS mold was able to drive a solution with a viscosity under 575 mPa·s. A simulation model was developed which can well estimate the flow rate in various dimensions of micro-channels. Cross-linked albumin has low viscosity (1 mPa·s) in aqueous solution and can undergo a one-way dehydration process from solution to solid that produces cellular repellency after dehydration. A microstructure of cross-linked albumin was fabricated on a cell culture dish by the microcasting method. After cells were seeded and cultivated on the cell culture dish with the microstructure for 7 days, the cellular pattern of mouse skeletal myoblast cell line C2C12 was observed. The microcasting with cross-linked albumin solution enables preparation of patterned cell culture systems more quickly in comparison with the previous agarose gel casting, which requires a gelation process before the dehydration process.

Vacuum microcasting of 2-methacryloyloxyethyl phosphorylcholine polymer for stable cell patterning.

This study demonstrates the rapid fabrication and utility of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer film for cell patterning. The film was obtained on a cell culture surface by microcasting MPC polymer ethanol solution into a degassed polydimethylsiloxane mold with a desired pattern. After removal of the mold, 293AD cells were cultured on the surface of the polymer film with the patterned microstructures. Patterned cell adhesion restricted by the film was successfully maintained during at least a 168-h cultivation. The microcast MPC polymer film can be prepared rapidly and used for efficient long-term cell confinement.

Demonstration of a bio-microactuator powered by vascular smooth muscle cells coupled to polymer micropillars.

We have demonstrated the working principle of a bio-microactuator using smooth muscle cells (SMCs) by driving micropillars coupled to cultured SMCs and controlled pillar displacements by chemical stimuli; the generated driving force was estimated to be over 1.1 microN.

A micro-spherical heart pump powered by cultured cardiomyocytes.

Miniaturization of chemical or biochemical systems creates extremely efficient devices exploiting the advantages of microspaces. Although they are often targeted for implanted tissue engineered organs or drug-delivery devices because of their highly integrated systems, microfluidic devices are usually powered by external energy sources and therefore difficult to be used in vivo. A microfluidic device powered without the need for external energy sources or stimuli is needed. Previously, we demonstrated the concept of a cardiomyocyte pump using only chemical energy input to cells as a driver (Yo Tanaka, Keisuke Morishima, Tatsuya Shimizu, Akihiko Kikuchi, Masayuki Yamato, Teruo Okano and Takehiko Kitamori, Lab Chip, 6(3), pp. 362-368). However, the structure of this prototype pump described there included complicated mechanical components and fabricated compartments. Here, we have created a micro-spherical heart-like pump powered by spontaneously contracting cardiomyocyte sheets driven without a need for external energy sources or coupled stimuli. This device was fabricated by wrapping a beating cardiomyocyte sheet exhibiting large contractile forces around a fabricated hollow elastomeric sphere (5 mm diameter, 250 microm polymer thickness) fixed with inlet and outlet ports. Fluid oscillations in a capillary connected to the hollow sphere induced by the synchronously pulsating cardiomyocyte sheet were confirmed, and the device continually worked for at least 5 days in this system. This bio/artificial hybrid fluidic pump device is innovative not only because it is driven by cells using only chemical energy input, but also because the design is an optimum structure (sphere). We anticipate that this device might be applied for various purposes including a bio-actuator for medical implant devices that relies on biochemical energy, not electrical interfacing.

Analysis of Long-term Morphological Changes of Micro-patterned Molecules and Cells on PDMS and Glass Surfaces.

We demonstrated that our previously developed gas-phase fluoroalkylsilane patterning method was applicable to polydimethylsiloxane (PDMS) and we compared the stability of patterned proteins and cultured cells between PDMS and glass surfaces. The shapes of the protein patterns were stable on both glass and PDMS surfaces for more than 1 week. The cell patterns were stable on glass surfaces for 1 week, while those on PDMS collapsed within a few days. These results indicated that our method was applicable to PDMS, although, compared with glass, PDMS has an unsolved issue for its application to long-term patterning of cells.

An actuated pump on-chip powered by cultured cardiomyocytes.

Cellular functions are frequently exploited as processing components for integrated chemical systems such as biochemical reactors and bioassay systems. Here, we have created a new cell-based microsystem exploiting the intrinsic pulsatile mechanical functions of cardiomyocytes to build a cellular micropump on-chip using cardiomyocyte sheets as prototype bio-microactuators. We first demonstrate cell-based control of fluid motion in a model microchannel without check valves and evaluate the potential performance of the bio-actuation. For this purpose, a poly(dimethylsiloxane) (PDMS) microchip with a microchannel equipped with a diaphragm and a push-bar structure capable of harnessing collective cell fluid mechanical forces was coupled to a cultured pulsating cardiomyocyte sheet, activating cell-based fluid movement in the microchannel by actuating the diaphragm. Cell oscillation frequency and correlated fluid displacement in this system depended on temperature. When culture temperature was increased, collective cell contraction frequency remained cooperative and synchronous but increased, while displacement was slightly reduced. We then demonstrated directional fluid pumping within microchannels using cantilever-type micro-check valves made of polyimide. A directional flow rate of nL min(-1) was produced. This cell micropump system could be further developed as a self-actuated and efficient mechanochemical transducer requiring no external energy sources for various purposes in the future.

Demonstration of a PDMS-based bio-microactuator using cultured cardiomyocytes to drive polymer micropillars.

Natural cellular functions are increasingly exploited for integrated chemical systems such as biochemical reactors and biosensors. We propose to utilize the intrinsic mechanical function of cardiomyocytes, converting chemical energy into mechanical energy. In this report, we demonstrate the working principle of our proposed poly(dimethylsiloxane) (PDMS) based cardiomyocyte bio-microactuator using fabricated PDMS micropillars driven to repetitive motion by attached pulsating cardiomyocytes. Sheets of PDMS embedded with an array of micropillars were fabricated and modified for cardiomyocyte attachment in culture. Primary neonatal rat cardiomyocytes were cultured on the array, attaching to the micropillars and substratum successfully, and exhibiting their typical spontaneous, pulsatile phenotype. Micropillars beat with the coupled cells spontaneously without any triggers. The beat frequency was 1.4 Hz at 37 degrees C and the displacement of the top of the pillar that beat most strongly in our observation was 2.8+/-0.2 microm. From this result, contractile forces of cultured cardiomyocytes were estimated to exceed 3.5 microN. The estimated force is far greater than that of a previously described hydrogel-based cardiomyocyte bio-microactuator (K. Morishima et al., in Micro Total Analysis Systems 2003, ed. M. A. Northrup et al., The Transducers Research Foundation, San Diego, CA, vol. 2, pp. 1125-1128). PDMS compatibility as a base material for bio-microactuator design using cultured cardiomyocytes was verified. This PDMS-based cell microactuator worked for about one week without exchange of the culture medium, and this system could be developed for various purposes in the future as self-actuated and efficient mechanochemical transducers without external energy source requirements.

Micro/nanoparticle separation via curved nano-gap device with enhanced size resolution.

Micro/nanoparticles are widely found in industry and biological field to play important roles and particle size distribution is an important factor to evaluate these particles. Nano-gap device has advantages in size determination for particles in diverse size and/or shape, but it has difficulty in practical use due to severe requirement on instrumental alignment to reproduce the gap profile and non-quantitative sample injection based on capillary action. To solve these problems, curved nano-gap device (CGD) was fabricated from two flat glass plates via a simple microfabrication process to gain enhanced size resolution, and pressure-driven liquid delivery system was coupled to CGD. The gap was precisely controlled by wet etching with hydrofluoric acid on a glass plate to obtain the depth of 35.5±15.0nm on average. CGD utilized glass deflection with 18.1nm elevation/µm lateral distance that achieved practical size resolutions of 14.5nm, which was 15.7% smaller than that of conventional linear nano-gap device. Using CGD, particles from 0.5 to 10µm diameter were trapped and separated. The estimated sizes of the trapped particles matched the suggested values well. Cell sizes were also measured by CGD and the measured values matched with the values found by microscope observation. CGD acquired reproducible instrumental setup that resulted in robust analysis on size of micro/nanoparticles.

Cultivation and recovery of vascular endothelial cells in microchannels of a separable micro-chemical chip.

Various micro cell culture systems have recently been developed. However, it is extremely difficult to recover cultured cells from a microchannel because the upper and lower substrates of a microchip are permanently combined. Therefore, we developed a cell culture and recovery system that uses a separable microchip with reversible combining that allows separation between closed and open channels. To realize this system, two problems related to microfluidic control-prevention of leakage and non-invasive recovery of cultured cells from the substrate-must be overcome. In the present study, we used surface chemistry modification to solve both problems. First, octadecyltrimethoxysilane (ODTMS) was utilized to control the Laplace pressure at the liquid/vapor phase interface, such that it was directed toward the microchannels, which suppressed leakage from the slight gap between two substrates. Second, a thermoresponsive polymer poly(N-isopropyl acrylamide) (PNIPAAm) was used to coat the surface of the ODTMS-modified microchannel by UV-mediated photopolymerization. PNIPAAm substrates are well known for controlled cell adhesion/detachment by alteration of temperature. Finally, the ODTMS- and PNIPAAm-modified separable microchips were subjected to patterning, and human arterial endothelial cells (HAECs) were cultured in the resulting microchannels with no leakage. After 96 h of the culture, the HAECs were detached from the microchips by decreasing the temperature and were then recovered from the microchannels. This study is the first to demonstrate the recovery of living cells cultured in a microchannel, and may be useful as a fundamental technique for vascular tissue engineering.

Basic structure and cell culture condition of a bioartificial renal tubule on chip towards a cell-based separation microdevice.

Various separation processes have been integrated in microfluidics, such as capillary electrophoresis and chromatography, on a microchip. However, it is extremely difficult to separate a complicated biological system by conventional methods. Here, we report on a feasible structure and the culture condition of human renal proximal tubule epithelial cells (RPTECs), with the aim to construct a bioartificial renal tubule on a chip. Glass microchips and a polycarbonate membrane were sealed with no leakage after a surface modification. Furthermore, matrigel was selected as an optimized extracellular matrix (ECM) for cell-proliferation on the membrane. After culturing for 5 days, RPTECs reached confluent in the chip-membrane structure, which was confirmed by nuclei staining. So far, we have constructed the basic structure and cell proliferation circumstance for the future demonstration of the RPTECs separating function. This separation microdevice has promising potential to be applied as both a unit of a circulation cell culture system and a research platform of cell biology.

Rapid screening swine foot-and-mouth disease virus using micro-ELISA system.

In order to tackle both regional and global foot-and-mouth disease virus (FMDV) epdimics, we hereby develop a rapid microfluidic thermal lens microscopic method to screen swine type O FMDV with good efficiency. The scheme has great merits in terms of field portability, sample volume, assay time, analytical sensitivity, and test reproducibility.