Cytoglobin functions as a redox regulator of melanogenesis in normal epidermal melanocytes.

Epidermal melanocytes are continuously exposed to sunlight-induced reactive oxygen species (ROS) and oxidative stress generated during the synthesis of melanin. Therefore, they have developed mechanisms that maintain normal redox homeostasis. Cytoglobin (CYGB), a ubiquitously expressed intracellular iron hexacoordinated globin, exhibits antioxidant activity and regulates the redox state of mammalian cells through its activities as peroxidase and nitric oxide (NO) dioxygenase. We postulated that CYGB functions in the melanogenic process as a regulator that maintains oxidative stress within a physiological level. This was examined by characterizing normal human melanocytes with the knockdown (KD) of CYGB using morphological and molecular biological criteria. CYGB-KD cells were larger, had more dendrites, and generated more melanin granules in the advanced stages of melanogenesis than control cells. The expression levels of major melanogenesis-associated genes and proteins were higher in CYGB-KD melanocytes than in wild type (WT) cells. As expected, CYGB-KD melanocytes generated more ROS and NO than WT cells. In conclusion, CYGB physiologically contributes to maintaining redox homeostasis in the melanogenic activity of normal melanocytes by controlling the intracellular levels of ROS and NO.

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.

A pressure driven electric energy generator exploiting a micro- to nano-scale glass porous filter with ion flow originating from water.

We demonstrated a pressure driven energy harvesting device using water and that features a glass filter with porous channels. We employed powder sintering to fabricate the glass filter (2 cm diameter, 3 mm thickness) by packing a powder of borosilicate glass particles into a carbon mold and then thermally fusing this at 700°C under pressure. In constant flow rate experiment, the optimum average pore radius of the filter for power generation was 12 µm. Using this filter, power of 3.8 mW (27 V, 0.14 mA, 0.021% energy efficiency) was generated at a water flow speed of 50 mm/s. In constant pressure experiment, a power generator was equipped with a foot press unit with a 60 kg weight (830 kPa) and 50 mL of water. The optimum average pore radius for power generation in this experiment was 12 µm and power of 4.8 mW (18 V, 0.26 mA, 0.017% energy efficiency) was generated with 1.7 s duration. This was enough power for direct LED lighting and the capacitors could store enough energy to rotate a fan and operate a wireless communicator. Our pressure driven device is suitable for energy harvesting from slow movements like certain human physiological functions, e.g. walking.

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.

Human iPS cell derived RPE strips for secure delivery of graft cells at a target place with minimal surgical invasion.

Several clinical studies have been conducted into the practicality and safety of regenerative therapy using hESC/iPSC-retinal pigment epithelium (RPE) as a treatment for the diseases including age-related macular degeneration. These studies used either suspensions of RPE cells or an RPE cell sheet. The cells can be injected using a minimally invasive procedure but the delivery of an intended number of cells at an exact target location is difficult; cell sheets take a longer time to prepare, and the surgical procedure is invasive but can be placed at the target area. In the research reported here, we combined the advantages of the two approaches by producing a quickly formed hiPSC-RPE strip in as short as 2 days. The strip readily expanded into a monolayer sheet on the plate, and after transplantation in nude rats, it showed a potency to partly expand with the correct apical/basal polarity in vivo, although limited in expansion area in the presence of healthy host RPE. The strip could be injected into a target area in animal eyes using a 24G canula tip.

Specific capture and intact release of breast cancer cells using a twin-layer vein-shaped microchip with a self-assembled surface.

Breast cancer is the most fatal disease among female cancers yet its detection still relies on needle biopsy. The unique physical and immune characteristics of breast cancer cells different from blood cells make them suitable to be employed as excellent biomarkers in liquid biopsy, through which breast cancer cells are collected from peripheral blood for further cancer diagnosis, medical treatment monitoring, and drug screening. Although the separation and enrichment of breast cancer cells from peripheral blood have been studied for years, there are still two problems to be solved in these methods: the low efficiency of on-chip immunologic capture in the flow state and the influence of the conjugated antibodies for the following analyses during cell release. In this paper, a vein-shaped microchip with self-assembled surface was developed for the specific and robust capture (91.2%) of breast cancer cells in the flow state. A protein-recovery process was proposed, in which trypsin served as a mild release reagent, releasing 92% of cells with high viability (96%), normal adherent proliferation, and complete proteins on the cell membrane, avoiding disturbance of the conjugated chemical molecules in the following clinical study. The excellent performance demonstrated in isolating free breast cancer cells from real peripheral blood sample, originating from the orthotopic 4T1 breast cancer metastatic models, suggest the microchip could be utilized as a multiple circulating tumor cell capture and release platform that could allow providing more reliable information in liquid biopsies.

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.

Hydrodynamic particle focusing enhanced by femtosecond laser deep grooving at low Reynolds numbers.

Microfluidic focusing of particles (both synthetic and biological), which enables precise control over the positions of particles in a tightly focused stream, is a prerequisite step for the downstream processing, such as detection, trapping and separation. In this study, we propose a novel hydrodynamic focusing method by taking advantage of open v-shaped microstructures on a glass substrate engraved by femtosecond pulse (fs) laser. The fs laser engraved microstructures were capable of focusing polystyrene particles and live cells in rectangular microchannels at relatively low Reynolds numbers (Re). Numerical simulations were performed to explain the mechanisms of particle focusing and experiments were carried out to investigate the effects of groove depth, groove number and flow rate on the performance of the groove-embedded microchannel for particle focusing. We found out that 10-µm polystyrene particles are directed toward the channel center under the effects of the groove-induced secondary flows in low-Re flows, e.g. Re < 1. Moreover, we achieved continuous focusing of live cells with different sizes ranging from 10 to 15 µm, i.e. human T-cell lymphoma Jurkat cells, rat adrenal pheochromocytoma PC12 cells and dog kidney MDCK cells. The glass grooves fabricated by fs laser are expected to be integrated with on-chip detection components, such as contact imaging and fluorescence lifetime-resolved imaging, for various biological and biomedical applications, where particle focusing at a relatively low flow rate is desirable.

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.

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.

Intelligent whole-blood imaging flow cytometry for simple, rapid, and cost-effective drug-susceptibility testing of leukemia.

Drug susceptibility (also called chemosensitivity) is an important criterion for developing a therapeutic strategy for various cancer types such as breast cancer and leukemia. Recently, functional assays such as high-content screening together with genomic analysis have been shown to be effective for predicting drug susceptibility, but their clinical applicability is poor since they are time-consuming (several days long), labor-intensive, and costly. Here we present a highly simple, rapid, and cost-effective liquid biopsy for ex vivo drug-susceptibility testing of leukemia. The method is based on an extreme-throughput (>1 million cells per second), label-free, whole-blood imaging flow cytometer with a deep convolutional autoencoder, enabling image-based identification of the drug susceptibility of every single white blood cell in whole blood within 24 hours by simply flowing a drug-treated whole blood sample as little as 500 µL into the imaging flow cytometer without labeling. Our results show that the method accurately evaluates the drug susceptibility of white blood cells from untreated patients with acute lymphoblastic leukemia. Our method holds promise for affordable precision medicine.

Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering.

Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

Time Sequential Single-Cell Patterning with High Efficiency and High Density.

Single-cell capture plays an important role in single-cell manipulation and analysis. This paper presents a microfluidic device for deterministic single-cell trapping based on the hydrodynamic trapping mechanism. The device is composed of an S-shaped loop channel and thousands of aligned trap units. This arrayed structure enables each row of the device to be treated equally and independently, as it has row periodicity. A theoretical model was established and a simulation was conducted to optimize the key geometric parameters, and the performance was evaluated by conducting experiments on MCF-7 and Jurkat cells. The results showed improvements in single-cell trapping ability, including loading efficiency, capture speed, and the density of the patterned cells. The optimized device can achieve a capture efficiency of up to 100% and single-cell capture efficiency of up to 95%. This device offers 200 trap units in an area of 1 mm², which enables 100 single cells to be observed simultaneously using a microscope with a 20× objective lens. One thousand cells can be trapped sequentially within 2 min; this is faster than the values obtained with previously reported devices. Furthermore, the cells can also be recovered by reversely infusing solutions. The structure can be easily extended to a large scale, and a patterned array with 32,000 trap sites was accomplished on a single chip. This device can be a powerful tool for high-throughput single-cell analysis, cell heterogeneity investigation, and drug screening.

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.

Optofluidic time-stretch quantitative phase microscopy.

Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective.

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.

Simple agarose micro-confinement array and machine-learning-based classification for analyzing the patterned differentiation of mesenchymal stem cells.

The geometrical confinement of small cell colonies gives differential cues to cells sitting at the periphery versus the core. To utilize this effect, for example to create spatially graded differentiation patterns of human mesenchymal stem cells (hMSCs) in vitro or to investigate underpinning mechanisms, the confinement needs to be robust for extended time periods. To create highly repeatable micro-fabricated structures for cellular patterning and high-throughput data mining, we employed here a simple casting method to fabricate more than 800 adhesive patches confined by agarose micro-walls. In addition, a machine learning based image processing software was developed (open code) to detect the differentiation patterns of the population of hMSCs automatically. Utilizing the agarose walls, the circular patterns of hMSCs were successfully maintained throughout 15 days of cell culture. After staining lipid droplets and alkaline phosphatase as the markers of adipogenic and osteogenic differentiation, respectively, the mega-pixels of RGB color images of hMSCs were processed by the software on a laptop PC within several minutes. The image analysis successfully showed that hMSCs sitting on the more central versus peripheral sections of the adhesive circles showed adipogenic versus osteogenic differentiation as reported previously, indicating the compatibility of patterned agarose walls to conventional microcontact printing. In addition, we found a considerable fraction of undifferentiated cells which are preferentially located at the peripheral part of the adhesive circles, even in differentiation-inducing culture media. In this study, we thus successfully demonstrated a simple framework for analyzing the patterned differentiation of hMSCs in confined microenvironments, which has a range of applications in biology, including stem cell biology.

Selective cell capture and analysis using shallow antibody-coated microchannels.

Demand for analysis of rare cells such as circulating tumor cells in blood at the single molecule level has recently grown. For this purpose, several cell separation methods based on antibody-coated micropillars have been developed (e.g., Nagrath et al., Nature 450, 1235-1239 (2007)). However, it is difficult to ensure capture of targeted cells by these methods because capture depends on the probability of cell-micropillar collisions. We developed a new structure that actively exploits cellular flexibility for more efficient capture of a small number of cells in a target area. The depth of the sandwiching channel was slightly smaller than the diameter of the cells to ensure contact with the channel wall. For cell selection, we used anti-epithelial cell adhesion molecule antibodies, which specifically bind epithelial cells. First, we demonstrated cell capture with human promyelocytic leukemia (HL-60) cells, which are relatively homogeneous in size; in situ single molecule analysis was verified by our rolling circle amplification (RCA) method. Then, we used breast cancer cells (SK-BR-3) in blood, and demonstrated selective capture and cancer marker (HER2) detection by RCA. Cell capture by antibody-coated microchannels was greater than with negative control cells (RPMI-1788 lymphocytes) and non-coated microchannels. This system can be used to analyze small numbers of target cells in large quantities of mixed samples.

Development of a microfluidic platform for single-cell secretion analysis using a direct photoactive cell-attaching method.

A precise understanding of individual cellular processes is essential to meet the expectations of most advanced cell biology. Therefore single-cell analysis is considered to be one of possible approach to overcome any misleading of cell characteristics by averaging large groups of cells in bulk conditions. In the present work, we modified a newly designed microchip for single-cell analysis and regulated the cell-adhesive area inside a cell-chamber of the microfluidic system. By using surface-modification techniques involving a silanization compound, a photo-labile linker and the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer were covalently bonded on the surface of a microchannel. The MPC polymer was utilized as a non-biofouling compound for inhibiting non-specific binding of the biological samples inside the microchannel, and was selectively removed by a photochemical reaction that controlled the cell attachment. To achieve the desired single-macrophage patterning and culture in the cell-chamber of the microchannel, the cell density and flow rate of the culture medium were optimized. We found that a cell density of 2.0 × 10(6) cells/ml was the appropriate condition to introduce a single cell in each cell chamber. Furthermore, the macrophage was cultured in a small size of the cell chamber in a safe way for 5 h at a flow rate of 0.2 µl/min under the medium condition. This strategy can be a powerful tool for broadening new possibilities in studies of individual cellular processes in a dynamic microfluidic device.