Microenvironmental Analysis and Control for Local Cells under Confluent Conditions via a Capillary-Based Microfluidic Device.

Sophisticated functions of biological tissues are supported by small biological units of cells that are localized within a region of 100 µm scale. The cells in these units secrete molecules to form their microenvironment to play a vital role in biological functions. Various microfluidic devices have been developed to analyze the microenvironment but were not designed for cells in a culture dish in a confluent condition, a typical setup for cell and tissue cultivation. This study presents a novel glass capillary-based microfluidic device for studying confluent cells in a culture dish. The multiple capillaries allow the device to confine the local flow in 100 µm or smaller scale to form two adjacent regions with different chemical properties; it can simultaneously perform local cell stimulation and collect secreted molecules from the stimulated cells. Cell removal was achieved upon trypsin stimulation from a limited area (3.8 × 10-3 ± 1.0 × 10-3 mm2), which corresponded to 7.6 ± 2.0 cells, using the mouse skeletal myoblast cell line (C2C12 cells) in a confluent condition. Microenvironmental analysis was demonstrated by measuring the secreted tumor necrosis factor alpha (TNF-α) collected from the microenvironment of the stimulated and unstimulated mouse leukemic monocyte cell line (RAW264 cells) to track temporal changes in the TNF-α production. The TNF-α secreted from stimulated cells was approximately four-fold higher than that from unstimulated cells in 90 min. This device enables local cell stimulation and the collection of secreted molecules for cells under confluent conditions, which contributes to the analysis of the cellular microenvironment.

Catalytic confinement effects in nanochannels: from biological synthesis to chemical engineering.

Catalytic reactions within nanochannels are of significant importance in disclosing the mechanisms of catalytic confinement effects and developing novel reaction systems for scientific and industrial demands. Interestingly, catalytic confinement effects exist in both biological and artificial nanochannels, which enhance the reaction performance of various chemical reactions. In this minireview, we investigate the recent advances on catalytic confinement effects in terms of the reactants, reaction processes, catalysts, and products in nanochannels. A systematic discussion of catalytic confinement effects associated with biological synthesis in bio-nanochannels and catalytic reactions in artificial nanochannels in chemical engineering is presented. Furthermore, we summarize the properties of reactions both in nature and chemical engineering and provide a brief overlook of this research field.

Anhydrobiotic chironomid larval motion-based multi-sensing microdevice for the exploration of survivable locations.

African chironomid (Polypedilum vanderplanki) larvae can suspend their metabolism by undergoing severe desiccation and then resume this activity by simple rehydration. We present a microdevice using interdigital comb electrodes to detect the larval motion using the natural surface charge of the living larvae in water. The larvae were most active 2 h after soaking them in water at 30°C; they exhibited motions with 2 Hz frequency. This was comparable to the signal obtained from the microdevice via fast Fourier transform (FFT) processing. The amplitude of the voltage and current were 0.11 mV and 730 nA, respectively. They would be enough to be detected by a low power consumption microcomputer. Temperature and pH sensing were demonstrated by detecting the vital motions of the revived larvae under different conditions. This multi-functional biosensor will be a useful microdevice to search for survivable locations under extreme environmental conditions like those on other planets.

Recent advances in microfluidic devices for single-cell cultivation: methods and applications.

Single-cell analysis is essential to improve our understanding of cell functionality from cellular and subcellular aspects for diagnosis and therapy. Single-cell cultivation is one of the most important processes in single-cell analysis, which allows the monitoring of actual information of individual cells and provides sufficient single-cell clones and cell-derived products for further analysis. The microfluidic device is a fast-rising system that offers efficient, effective, and sensitive single-cell cultivation and real-time single-cell analysis conducted either on-chip or off-chip. Here, we introduce the importance of single-cell cultivation from the aspects of cellular and subcellular studies. We highlight the materials and structures utilized in microfluidic devices for single-cell cultivation. We further discuss biological applications utilizing single-cell cultivation-based microfluidics, such as cellular phenotyping, cell-cell interactions, and omics profiling. Finally, present limitations and future prospects of microfluidics for single-cell cultivation are also discussed.

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.

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.

Area cooling enables thermal positioning and manipulation of single cells.

Contactless particle manipulation based on a thermal field has shown great potential for biological, medical, and materials science applications. However, thermal diffusion from a high-temperature area causes thermal damage to bio-samples. Besides, the permanent bonding of a sample chamber onto microheater substrates requires that the thermal field devices be non-disposable. These limitations impede use of the thermal manipulation approach. Here, a novel manipulation platform is proposed that combines microheaters and an area cooling system to produce enough force to steer sedimentary particles or cells and to limit the thermal diffusion. It uses the one-time fabricated motherboard and an exchangeable sample chamber that provides disposable use. Sedimentary objects can be steered to the bottom center of the thermal field by combined thermal convection and thermophoresis. Single particle or cell manipulation is realized by applying multiple microheaters in the platform. Results of a cell viability test confirmed the method's compatibility in biology fields. With its advantages of biocompatibility for live cells, operability for different sizes of particles and flexibility of platform fabrication, this novel manipulation platform has a high potential to become a powerful tool for biology research.

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.

A Microfluidic Platform Based on Robust Gas and Liquid Exchange for Long-term Culturing of Explanted Tissues.

Microfluidic devices are important platforms to culture and observe biological tissues. Compared with conventional setups, microfluidic devices have advantages in perfusion, including an enhanced delivery of nutrients and gases to tissues. However, explanted tissues can maintain their functions for only hours to days in microfluidic devices, although their observations are desired for weeks. The suprachiasmatic nucleus (SCN) is a brain region composed of heterogeneous cells to control the biological clock system through synchronizing individual cells in this region. The synchronized and complicated cell-cell interactions of SCN cells are difficult to reproduce from seeded cells. Thus, the viability of explanted SCN contributes to the study of SCN functions. In this paper, we propose a new perfusion platform combining a PDMS microfluidic device with a porous membrane to culture an explanted SCN for 25 days. We expect that this platform will provide a universal interface for microfluidic manipulation of tissue explants.

Simple Isolation of Single Cell: Thin Glass Microfluidic Device for Observation of Isolated Single Euglena gracilis Cells.

Single cell analysis has gained attention as a means to investigate the heterogeneity of cells and amplify a cell with desired characteristics. However, obtaining a single cell from a large number of cells remains difficult because preparation of single-cell samples relies on conventional techniques such as pipetting that are labor intensive. In this study, we developed a system combining a 0.6-mm thin glass microfluidic device and machine vision approach to isolate single Euglena gracilis cells, as a model of microorganism with mobility, in a small/thin glass chamber. A single E. gracilis cell in a chamber was cultured for 4 days to monitor its multiplication. With this system, we successfully simplified preparation of single cells of interest and determined that it is possible to combine it with other analytical techniques to observe single cells continuously.