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.

Bio-actuated microvalve in microfluidics using sensing and actuating function of Mimosa pudica.

Bio-actuators and sensors are increasingly employed in microscale devices for numerous applications. Unlike other artificial devices actuated by living cells or tissues, here we introduce a microvalve system actuated by the stimuli-responsive action plant, Mimosa pudica (sleepy plant). This system realizes the control of the valve to open and close by dropping and recovering responses of Mimosa pudica branch upon external physical stimulations. The results showed that one matured single uncut Mimosa pudica branch produced average force of 15.82 ± 0.7 mN. This force was sufficient for actuating and keeping the valve open for 8.46 ± 1.33 min in a stimulation-recovering cycle of 30 min. Additionally, two separately cut Mimosa pudica branches were able to keep the valve open for 2.28 ± 0.63 min in a stimulating-recovering cycle of 20min. The pressure resistance and the response time of the valve were 4.2 kPa and 1.4 s, respectively. This demonstration of plant-microfluidics integration encourages exploiting more applications of microfluidic platforms that involve plant science and plant energy harvesting.

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.

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.

A sub-population of Dictyostelium discoideum cells shows extremely high sensitivity to cAMP for directional migration.

The chemotaxis of Dictysotelium discoideum cells in response to a chemical gradient of cyclic adenosine 3',5'-monophosphate (cAMP) was studied using a newly designed microfluidic device. The device consists of 800 cell-sized channels in parallel, each 4 µm wide, 5 µm high, and 100 µm long, allowing us to prepare the same chemical gradient in all channels and observe the motility of 500-1000 individual cells simultaneously. The percentage of cells that exhibited directed migration was determined for various cAMP concentrations ranging from 0.1 pM to 10 µM. The results show that chemotaxis was highest at 100 nM cAMP, consistent with previous observations. At concentrations as low as 10 pM, about 16% of cells still exhibited chemotaxis, suggesting that the receptor occupancy of only 6 cAMP molecules/cell can induce chemotaxis in very sensitive cells. At 100 pM cAMP, chemotaxis was suppressed due to the self-production and secretion of intracellular cAMP induced by extracellular cAMP. Overall, systematic observations of a large number of individual cells under the same chemical gradients revealed the heterogeneity of chemotaxis responses in a genetically homogeneous cell population, especially the existence of a sub-population with extremely high sensitivity for chemotaxis.

Effects of Flow-Induced Microfluidic Chip Wall Deformation on Imaging Flow Cytometry.

Imaging flow cytometry is a powerful tool by virtue of its capability for high-throughput cell analysis. The advent of high-speed optical imaging methods on a microfluidic platform has significantly improved cell throughput and brought many degrees of freedom to instrumentation and applications over the last decade, but it also poses a predicament on microfluidic chips. Specifically, as the throughput increases, the flow speed also increases (currently reaching 10 m/s): consequently, the increased hydrodynamic pressure on the microfluidic chip deforms the wall of the microchannel and produces detrimental effects lead to defocused and blur image. Here, we present a comprehensive study of the effects of flow-induced microfluidic chip wall deformation on imaging flow cytometry. We fabricated three types of microfluidic chips with the same geometry and different degrees of stiffness made of polydimethylsiloxane (PDMS) and glass to investigate material influence on image quality. First, we found the maximum deformation of a PDMS microchannel was >60 µm at a pressure of 0.6 MPa, while no appreciable deformation was identified in a glass microchannel at the same pressure. Second, we found the deviation of lag time that indicating velocity difference of migrating microbeads due to the deformation of the microchannel was 29.3 ms in a PDMS microchannel and 14.9 ms in a glass microchannel. Third, the glass microchannel focused cells into a slightly narrower stream in the X-Y plane and a significantly narrower stream in the Z-axis direction (focusing percentages were increased 30%, 32%, and 5.7% in the glass channel at flow velocities of 0.5, 1.5, and 3 m/s, respectively), and the glass microchannel showed stabler equilibrium positions of focused cells regardless of flow velocity. Finally, we achieved the world's fastest imaging flow cytometry by combining a glass microfluidic device with an optofluidic time-stretch microscopy imaging technique at a flow velocity of 25 m/s. © 2019 International Society for Advancement of Cytometry.

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.

Ultrasensitive Single Cell Metabolomics by Capillary Electrophoresis-Mass Spectrometry with a Thin-Walled Tapered Emitter and Large-Volume Dual Sample Preconcentration.

Single cell metabolome analysis is essential for studying microscale life phenomena such as neuronal networks and tumor microenvironments. Capillary electrophoresis-mass spectrometry (CE-MS) is one of the most sensitive technologies; however, its sensitivity is still not enough for single cell analysis on general human cells such as HeLa. To address these issues, we first developed an efficient ionization emitter, named as a "nanoCESI" emitter, that had a thin-walled (∼10 µm) and tapered (5-10 µm) end. The thin conductive wall enabled sheathless ionization and minimized the flow rate of ionizing sample, and the tapered end efficiently ionized analytes via an electrospray ionization mechanism, providing up to 3.5-fold increase in sensitivity compared with a conventional sheathless emitter. Fifty repetitive analyses on 20 amino acids were successfully achieved with a nanoCESI emitter. Relative standard deviations of 50 analyses were 1.5%, 4.4%, and 6.8% for migration time, peak height, and peak area, respectively, where a limit of detection (LOD) of 170 pM (850 zmol) was achieved. Second, a sample enrichment method, large-volume dual preconcentration by isotachophoresis and stacking (LDIS), was applied to a newly designed protocol of nanoCESI-MS. This approach achieved up to 380-fold enhanced sensitivity and LOD of 450 fM. Compared with normal sheathless CE-MS, coupling of nanoCESI and LDIS provided up to 800-fold increase of sensitivity in total. Finally, metabolome analyses of single HeLa cells were performed, where 20 amino acids were successfully quantified with triple-quadrupole MS and 40 metabolites were identified with quadrupole-time-of-flight MS, as a promising analytical platform for microscale bioanalysis for the next generation.

Isolating Single Euglena gracilis Cells by Glass Microfluidics for Raman Analysis of Paramylon Biogenesis.

Time-course analysis of single cells is important to characterize heterogeneous activities of individual cells such as the metabolic response to their environment. Single-cell isolation is an essential step prior to time-course analysis of individual cells by collecting, culturing, and identifying multiple single-cell targets. Although single-cell isolation has been performed by various methods previously, a glass microfluidic device with semiclosed microchannels dramatically improved this process with its simple operation and easy transfer for time-course analysis of identified single cells. This study demonstrates isolating single cells of the highly motile microalgae, Euglena gracilis, by semiclosed microchannels with liquid flow only. The isolated single cells were identified in isolating channels and continuously cultured to track, by Raman microscopy, for the formation of subcellular granules composed of polysaccharide paramylon, a unique metabolite of E. gracilis, generated through photosynthesis. Through low-temperature glass bonding, a thin glass interface was incorporated to the microfluidic device. Thus, the device could perform the direct measurements of cultured single cells at high magnification by Raman microscopy with low background noise. In this study, the first demonstration of sequential monitoring of paramylon biogenesis in a single identified E. gracilis cell is shown.

Enhancement in acoustic focusing of micro and nanoparticles by thinning a microfluidic device.

The manipulation of micro/nanoparticles has become increasingly important in biological and industrial fields. As a non-contact method for particle manipulation, acoustic focusing has been applied in sorting, enrichment and analysis of particles with microfluidic devices. Although the frequency and amplitude of acoustic waves and the dimensions of microchannels have been recognized as important parameters for acoustic focusing, the thickness of microfluidic devices has not been considered so far. Here, we report that thin glass microfluidic devices enhance acoustic focusing of micro/nanoparticles. It was found that the thickness of a microfluidic device strongly influences its ability to focus particles via acoustic radiation, because the energy propagation of acoustic waves is affected by the total mass of the device. Acoustic focusing of submicrometre polystyrene beads and Escherichia coli as well as enrichment of polystyrene beads were achieved in glass microfluidic devices as thin as 0.4 mm. Modifying the thickness of a microfluidic device can thus serve as a critical parameter for acoustic focusing when conventional parameters to achieve this effect are kept unchanged. Thus, our findings enable new approaches to the design of novel microfluidic devices.

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.

Profiling of N-linked glycans from 100 cells by capillary electrophoresis with large-volume dual preconcentration by isotachophoresis and stacking.

Glycan structure is changed in response with pathogenesis like cancer. Profiling of glycans from limited number of pathogenetic cells in an early-stage tissue is essential for discovering effective drugs. For analyzing tiny biological samples, we developed sensitive, high-resolution, and salt-tolerant method for analyzing trace level of N-linked glycans by coupling capillary electrophoresis (CE), laser-induced fluorescence (LIF) detection, and a new online sample preconcentration (OSP) method named "large-volume dual preconcentration by isotachophoresis and stacking (LDIS)", which is composed of two OSP methods, large-volume sample stacking (LVSS) and transient isotachophoresis (tITP). A typical LDIS-CE-LIF protocol was simple: a short-plug of leading electrolyte (LE) and large-volume sample solution were introduced to a capillary, followed by application of constant voltage. In the analysis of glucose ladder labeled with 8-aminopyrene-1,3,6-trisulfonic acid with 10 mM sodium chloride as LE, up to 2300-fold sensitivity increase was achieved with higher resolution than those in normal CE. By applying pressure assist during preconcentration, both viscous gel electrolyte and salty matrix of up to 10 mM NaCl were acceptable. Finally, N-glycans from approximately 100 cells (HeLa, MCF7, and HepG2) were analyzed as the model of localized tumor cells. From 30 to 40 glycans were successfully detected with almost same profile of large-scale sample. N-glycan structure could be predicted by searching glucose-unit value via Glycobase database, indicating that HepG2 expressed more sialylated glycans and MCF-7 expressed less glycans respectively, comparing with HeLa cells. It suggests the potential of LDIS-CE-LIF for discovery of disease-specific N-linked glycans in microscale environment.

A method of packaging molecule/cell-patterns in an open space into a glass microfluidic channel by combining pressure-based low/room temperature bonding and fluorosilane patterning.

We report a fabrication method of a "post-molecule/cell patterned" glass microchip using pressure-based low/room temperature bonding in dry conditions combined with fluorosilane patterning. Multiple proteins/cells were patterned in a single channel using this method. This simple method will provide benefits for using microchips for high throughput analysis in many biological experiments.

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.

D-Alanine in the islets of Langerhans of rat pancreas.

Relatively high levels of D-alanine (D-Ala), an endogenous D-amino acid, have been found in the endocrine systems of several animals, especially in the anterior pituitary; however, its functional importance remains largely unknown. We observed D-Ala in islets of Langerhans isolated from rat pancreas in significantly higher levels than in the anterior/intermediate pituitary; specifically, 180±60 fmol D-Ala per islet (300±100 nmol/gislet), and 10±2.5 nmol/g of wet tissue in pituitary. Additionally, 12±5% of the free Ala in the islets was in the d form, almost an order of magnitude higher than the percentage of D-Ala found in the pituitary. Surprisingly, glucose stimulation of the islets resulted in D-Ala release of 0.6±0.5 fmol per islet. As D-Ala is stored in islets and released in response to changes in extracellular glucose, D-Ala may have a hormonal role.

Signals from the brainstem sleep/wake centers regulate behavioral timing via the circadian clock.

Sleep-wake cycling is controlled by the complex interplay between two brain systems, one which controls vigilance state, regulating the transition between sleep and wake, and the other circadian, which communicates time-of-day. Together, they align sleep appropriately with energetic need and the day-night cycle. Neural circuits connect brain stem sites that regulate vigilance state with the suprachiasmatic nucleus (SCN), the master circadian clock, but the function of these connections has been unknown. Coupling discrete stimulation of pontine nuclei controlling vigilance state with analytical chemical measurements of intra-SCN microdialysates in mouse, we found significant neurotransmitter release at the SCN and, concomitantly, resetting of behavioral circadian rhythms. Depending upon stimulus conditions and time-of-day, SCN acetylcholine and/or glutamate levels were augmented and generated shifts of behavioral rhythms. These results establish modes of neurochemical communication from brain regions controlling vigilance state to the central circadian clock, with behavioral consequences. They suggest a basis for dynamic integration across brain systems that regulate vigilance states, and a potential vulnerability to altered communication in sleep disorders.

D-Aspartate acts as a signaling molecule in nervous and neuroendocrine systems.

D-Aspartate (D-Asp) is an endogenous amino acid in the central nervous and reproductive systems of vertebrates and invertebrates. High concentrations of D-Asp are found in distinct anatomical locations, suggesting that it has specific physiological roles in animals. Many of the characteristics of D-Asp have been documented, including its tissue and cellular distribution, formation and degradation, as well as the responses elicited by D-Asp application. D-Asp performs important roles related to nervous system development and hormone regulation; in addition, it appears to act as a cell-to-cell signaling molecule. Recent studies have shown that D-Asp fulfills many, if not all, of the definitions of a classical neurotransmitter-that the molecule's biosynthesis, degradation, uptake, and release take place within the presynaptic neuron, and that it triggers a response in the postsynaptic neuron after its release. Accumulating evidence suggests that these criteria are met by a heterogeneous distribution of enzymes for D-Asp's biosynthesis and degradation, an appropriate uptake mechanism, localization within synaptic vesicles, and a postsynaptic response via an ionotropic receptor. Although D-Asp receptors remain to be characterized, the postsynaptic response of D-Asp has been studied and several L-glutamate receptors are known to respond to D-Asp. In this review, we discuss the current status of research on D-Asp in neuronal and neuroendocrine systems, and highlight results that support D-Asp's role as a signaling molecule.

A novel pyridoxal 5'-phosphate-dependent amino acid racemase in the Aplysia californica central nervous system.

D-aspartate (D-Asp) is found in specific neurons, transported to neuronal terminals and released in a stimulation-dependent manner. Because D-Asp formation is not well understood, determining its function has proved challenging. Significant levels of D-Asp are present in the cerebral ganglion of the F- and C-clusters of the invertebrate Aplysia californica, and D-Asp appears to be involved in cell-cell communication in this system. Here, we describe a novel protein, DAR1, from A. californica that can convert aspartate and serine to their other chiral form in a pyridoxal 5'-phosphate (PLP)-dependent manner. DAR1 has a predicted length of 325 amino acids and is 55% identical to the bivalve aspartate racemase, EC 5.1.1.13, and 41% identical to the mammalian serine racemase, EC 5.1.1.18. However, it is only 14% identical to the recently reported mammalian aspartate racemase, DR, which is closely related to glutamate-oxaloacetate transaminase, EC 2.6.1.1. Using whole-mount immunohistochemistry staining of the A. californica central nervous system, we localized DAR1-like immunoreactivity to the medial region of the cerebral ganglion where the F- and C-clusters are situated. The biochemical and functional similarities between DAR1 and other animal serine and aspartate racemases make it valuable for examining PLP-dependent racemases, promising to increase our knowledge of enzyme regulation and ultimately, D-serine and D-Asp signaling pathways.

Photochemical Smiles rearrangement and Meisenheimer complex formation catalyzed by hydroxide ion via electron hole transfer catalysis.

[reactions: see text] Photochemical para-to-nitro Smiles rearrangement and para-to-nitro Meisenheimer complex formation occurs for nitrophenoxyethylamines with high concentrations of hydroxide ion in aqueous solution. Both photoreactions show first-order dependence on hydroxide ion concentration, but the mechanism involving hydroxide ion does not involve acid-base catalysis. The reactions take place from the triplet excited states of the nitrophenyl ethers. Analysis of quantum yields and kinetics is consistent with an electron hole transfer catalysis mechanism.