鈴木 雅登

ABSTRACT We developed a novel electrorotation (ROT) device featuring a microwell array with three electrodes. This device allows to monitor the increase in membrane capacitance of cells subjected to chemical stimulation. The microwell array is integrated into the bottom of a fluidic channel and holds rotating cells during stimulation with a solution containing a chemical agent. Positive dielectrophoresis (p‐DEP) effectively traps cells in microwells, whereas negative DEP (n‐DEP) facilitates the rapid formation of single‐cell presence. Alternating current (AC) voltages with a 120° phase shift applied across the three electrodes enable vertical and simultaneous rotation of cells. We observed a peak in rotation rate as a function of applied frequency, with the frequency spectrum shifting to lower frequencies as membrane capacitance increased. A positive correlation was identified between rotation rate and membrane capacitance, so monitoring in the low‐frequency range is advantageous. Although n‐DEP at lower frequencies risks removing cells from microwells, the continuous monitoring of the ROT rate during chemical stimulation was achieved by regulating the height of the ROT center of cells. We demonstrated the monitoring of membrane capacitance increase induced by Ca²⁺ influx from ionomycin. This simple configuration facilitates statistical analysis of ROT rates without fluorescent labeling, making it suitable for label‐free assessments of white blood cells’ responses to stimuli.

Many sexually mature women experience premenstrual syndrome (PMS) or premenstrual dysphoric mood disorder (PMDD). Current approaches for managing PMS and PMDD rely on daily mental condition recording, which many discontinue due to its impracticality. Hence, there's a critical need for a simple, objective method to monitor mental symptoms. One of the principal symptoms of PMDD is a dysfunction in emotional regulation, which has been demonstrated through brain-function imaging measurements to involve hyperactivity in the amygdala and a decrease in functionality in the prefrontal cortex (PFC). However, most research has been focused on PMDD, leaving a gap in understanding of PMS. Near-infrared spectroscopy (NIRS) measures brain activity by spectroscopically determining the amount of hemoglobin in the blood vessels. This study aimed to characterize the emotional regulation function in PMS. We measured brain activity in the PFC region using NIRS when participants were presented with emotion-inducing pictures. Furthermore, moods highly associated with emotions were assessed through questionnaires. Forty-six participants were categorized into non-PMS, PMS, and PMDD groups based on the gynecologist's diagnosis. POMS2 scores revealed higher negative mood and lower positive mood in the follicular phase for the PMS group, while the PMDD group exhibited heightened negative mood during the luteal phase. NIRS results showed reduced emotional expression in the PMS group during both phases, while no significant differences were observed in the PMDD group compared to non-PMS. It was found that there are differences in the distribution of mood during the luteal and follicular phase and in cerebral blood flow responses to emotional stimuli between PMS and PMDD. These findings suggest the potential for providing individuals with awareness of PMS or PMDD through scores on the POMS2 and NIRS measurements.

Many sexually mature females suffer from premenstrual syndrome (PMS), but effective coping methods for PMS are limited due to the complexity of symptoms and unclear pathogenesis. Awareness has shown promise in alleviating PMS symptoms but faces challenges in long-term recording and consistency. Our research goal is to establish a convenient and simple method to make individual female aware of their own psychological, and autonomic conditions. In previous research, we demonstrated that participants could be classified into non-PMS and PMS groups based on mood scores obtained during the follicular phase. However, the properties of neurophysiological activity in the participants classified by mood scores have not been elucidated. This study aimed to classify participants based on their scores on a mood questionnaire during the follicular phase and to evaluate their autonomic nervous system (ANS) activity using a simple device that measures pulse waves from the earlobe. Participants were grouped into Cluster I (high positive mood) and Cluster II (low mood). Cluster II participants showed reduced parasympathetic nervous system activity from the follicular to the menstrual phase, indicating potential PMS symptoms. The study demonstrates the feasibility of using mood scores to classify individuals into PMS and non-PMS groups and monitor ANS changes across menstrual phases. Despite limitations such as sample size and device variability, the findings highlight a promising avenue for convenient PMS self-monitoring.

Simultaneous electrorotation in microwells during chemical stimulation label-free monitoring effect of chemicals in single-cell manner.

Introduction: We have developed manipulation techniques to form cell-based arrays by positive dielectrophoresis (p-DEP) and to retrieve target cells from cell−based arrays selectively by negative dielectrophoresis (n-DEP). The novel devices with microwell arrays on microband electrodes were employed to manipulate cells. Hybridomas with the secretion ability of antibodies were trapped to form cell-based arrays. Then, the ability was discriminated by trapping the antibodies to the antigen immobilized on the bottom of wells. Finally, individual cells trapped in microwells were selectively retrieved by regulating the generation of electric fields in individual microwells. The development of series of these techniques could be useful to recover hybridomas producing antibodies with high affinity in large populations of cells without repeated steps of a culture and a limiting serial-dilution. Experimental: The device comprises the upper substrate with indium-tin-oxide (ITO) microband electrodes and the lower substrate with microwell arrays on ITO microband electrodes. Hybridomas producing an anti-rabbit serum albumin (RSA) antibody suspended in the DEP medium were introduced in the channel. AC signal (3 MHz, 2 Vpp) was then applied to the upper and lower microband electrodes with opposite phase to form a cell-based array. Anti-RSA antibodies secreted from hybridomas trapped in the microwells were captured by RSA immobilized on the electrodes. Cell arrays were then treated with anti-mouse IgG antibody conjugated with Alexa 488 (a secondary antibody). Thereafter, a repulsive force of negative dielectrophoresis (n-DEP) was employed to retrieve the target hybridomas from the microwell array. To retrieve the target hybridoma, an AC signal in the n-DEP frequency region was applied to a pair of microband electrodes above and below the microwell with the target hybridoma. Results and discussion: Cell−based arrays were formed with the occupancy efficiency of over 90% in a few seconds by p-DEP. Antibodies secreted in microwell arrays were captured to discriminate target hybridomas in a few hours without repeated steps of a culture and a limiting serial-dilution. Furthermore, hybridomas trapped in microwells were retrieved from the cell−based array by applying an AC signal to band electrodes. The sequential system for forming cell−based arrays, discriminating hybridomas secreting specific antibodies, and retrieving target hybridomas was developed by using the novel microwell array device comprising 3-D microband array electrodes with an orthogonal arrangement.

Introduction Electrical properties of cells (i.e. membrane capacitance and cytoplasm conductivity) have been studied to understand the complex physiological states of cells and used as markers for determining cell types. Electrorotation (ROT) that is one of the alternating current electrokinetic phenomena has been utilized to characterize the electrical properties of single cells. In ROT techniques, quadrupole electrodes were conventionally employed to induce a rotating electric field at the center of them by applying sine waves with 90° differences of each phase. However, a relatively long experimental period was required because several single cells must be repeatedly arranged to a center of quadrupole electrodes. In this presentation, we demonstrated the simultaneous ROT measurement of K562 cells by using the 3D-interdigitated array (3D-IDA) device to determine the membrane capacitances and cytoplasm conductivities of the different types of cells [1]. The rotating electric fields in over 1,500 grids were simultaneously generated by this device to obtain the distribution of rotation rates of over 50 cells with single operation [2]. The grid is defined as regions surrounded by four microband electrodes. We have also developed a new 3D-IDA device with microwells to maintain the cells in them during a chemical stimulation by a fluid flow. We investigated ROT rates accompanied with the changes of the dielectric properties of cells during the stimulation with an activator regent for Jurkat cells. This is the first report to monitor the changes of ROT rates upon chemical stimuli to investigate the effect of chemical agents to cellular function. Experimental Methods Construction of 3D-IDA device The device consisted of two IDA electrodes (20 μm in width, 30 μm gaps) made from indium tin-oxide (ITO). The IDA electrode was overlaid on another IDA electrode by an orthogonal arrangement via a double-sided adhesive film (30 μm in thickness) (Fig. A). A constant rotational electric field was generated in the grids by applying AC voltages with 90° differences of each phase to four microband electrodes. Construction of 3D-IDA device with microwells Microwells with rectangle shape (26 mm in long side and 20 mm in short side) were fabricated with an insulative photoresist (20 mm in thickness) on the gaps of ITO-IDA electrodes. Two ITO-electrodes (3 µm × 20 µm) were arranged at both short sides on the bottoms of microwells. Two gold electrodes (3 µm × 20 µm) were also arranged at both long sides on the insulative photoresist (Fig. B). Results and Conclusions Simultaneous ROT measurement of K562 cells using the 3D-IDA device K562 cells were resuspended in a ROT solution with the conductivity of 360 mS m⁻¹ (the mixture of 75% (v/v) of 300 mM mannitol and 25% (v/v) of RPMI 1640 medium) and subjected to ROT measurement by 3D-IDA devices. When AC signals (700 kHz, 5 Vpp) were applied, K562 cells dispersed randomly started to rotate and move to the center of each grid. The ratio of the grids occupied with single cells was 45%, when the cell suspension with the concentration of 4.0 × 10⁷ cells mL-1 was injected (Fig, A). Thus, we could obtain the rotation rates of 450–680 cells (40–60 cells in single images) simultaneously and estimated the ROT rate as 8.53 radian s^-1(Fig. C). Monitoring of ROT behavior of single cells stimulated with calcium ionophore The ROT device with the microwells were filled with Jurkat cells resuspended in the ROT solution adjusted to 5 × 10⁵ cells mL^-1 and incubated for 2.5 min to arrange cells in the microwells. The ratio of microwells occupied with single cells were 27% (Fig. B). When AC voltages (2 Vpp, 300 kHz) with different phases were applied to four electrodes, the cells began to rotate (Fig. D). The average ROT rate of Jurkat cells was estimated and found to be 1.01 radian s^-1. Then, the solution containing 1 mM ionomycin which is well known as an activator for Jurkat cells to lead to the decrease of the membrane capacitance [3] was injected in the device. Although the solution above the microwells was disturbed by the fluidic flow (40 mm s^-1), the cells rotated with the constant rate in the microwells. The ROT rate gradually decreased and reached at 0.73 radian s^-1 40 s after the injection. The result could reflect the decrease of the membrane capacitance due to the activation of Jurkat cells induced by the stimulus of ionomycin. The 3D-IDA device with microwells would be applied to the high through-put screening of drugs and discrimination of highly responsible cells to drugs. References (1) Ino, K.; Ishida, A.; Inoue, K. Y.; Suzuki, M.; Koide, M.; Yasukawa, T.; Shiku, H.; Matsue, T. Electrorotation Chip Consisting of Three-Dimensional Interdigitated Array Electrodes. Sensors Actuators, B Chem.2011, 153 (2), 468–473. https://doi.org/10.1016/j.snb.2010.11.012. (2) Kawai, S.; Suzuki, M.; Arimoto, S.; Korenaga, T.; Yasukawa, T. Determination of Membrane Capacitance and Cytoplasm Conductivity by Simultaneous Electrorotation. Analyst2020, 145 (12), 4188–4195. https://doi.org/10.1039/d0an00100g. (3) Pethig, R.; Bressler, V.; Carswell‐Crumpton, C.; Chen, Y.; Foster‐Haje, L.; García‐Ojeda, M. E.; Lee, R. S.; Lock, G. M.; Talary, M. S.; Tate, K. M. Dielectrophoretic Studies of the Activation of Human T Lymphocytes Using a Newly Developed Cell Profiling System. Electrophoresis2002, 23 (13), 2057–2063. https://doi.org/10.1002/1522-2683(200207)23:13&lt;2057::AID-ELPS2057&gt;3.0.CO;2-X. Figure 1 <p></p>

Introduction Cell microarrays which sectionalized cells into microwells are powerful tool for elucidating exhaustively functions of cells at single cell level. However, there are some difficulties in taking advantages of the cell array. At first, the “single-cell occupancy”, which is the ratio of microwells containing one cell to total microwell, is low. Typically, the sedimentation of cells with their own weight was used to form cell array, resulting in a low “single-cell occupancy” as low as about 50%. The second difficulty is that it takes time and effort to retrieve the target cell due to the precise and careful manipulation of the micropippet at resolution of micrometer. The broad generalization of cell microarrays requires the development of techniques to efficiently fabricate cell arrays and to easily pick up cells from cell populations. Dielectrophoresis (DEP) has become attractive because it allows for easy, rapid, and mass manipulation of cells. We have previously demonstrated the dielectrophoretic trapping cells in microwells within a few seconds with an occupation efficiency over 95% using a microwell array device. The device was fabricated by placing the upper indium-tin oxide (ITO) substrate on top of the bottom ITO electrode covered with an insulating layer with a microwell pattern. Although this device has the advantage of producing cell arrays rapidly at high density, it did not selectively manipulate the cells on the microwells. In this study, we propose a simple device for flexible dielectrophoretic manipulation of cells based on the combination of positive DEP (p-DEP) and negative DEP (n-DEP). The use of the present device allows for accurate retrieval of the target cells from a microwell array with retaining the undesired on microwells, not to mention the forming of cell arrays. This device was comprised an upper substrate with microband electrodes mounted on a lower substrate with microwells on the same design of microband electrodes by 90 degree relative to the bottom substrate (Fig. A). The layout of electrodes enables to retrieve the target cell by the repulsive force of n-DEP induced by applying an AC voltage to two microband electrodes arranged above and below the microwell containing the target cell. Naturally, cell arrays can also be fabricated by the attractive force to bottom of microwell induced with p-DEP by applying an AC voltage to all microband electrodes. Experiments and Results The upper and lower ITO substrates with patterns of microband array were fabricated by a conventional photolithographic method. Width of the microband electrode and gap between the electrodes were 40 µm and 80 µm, respectively. The pattern of microwell (16 µm in diameter and 10 µm in height) on the microband electrodes on the lower substate was made of a negative photoresist SU-8. Microband array electrodes on the upper substrate were located 30 µm above the microwells on the lower substrate to form 144 intersections comprising microband electrodes containing microwells. Cell mixtures (3.0 × 10⁶ cells/mL) of hybridoma stained in green and red were prepared in a ratio of 10:1 to demonstrate the retrieval of red cells designated as the target cell. The application of the AC signal (1 MHz, 3 Vpp) to both the upper and the lower microband electrodes with opposite phasing resulted in the formation of the cell array. A single red cell was trapped in the 1–G well and eight green cells were trapped in the others (Fig. B). Subsequently, the frequency applied to band electrode 1 on the upper substrate and the band electrode G on the lower substrate was switched from 1 MHz for p-DEP to 300 kHz for n-DEP, while the frequency applied to the other band electrodes was maintained at 1 MHz for p-DEP (Fig. C). The target red cell in 1–G well was gradually removed over a few seconds after switching the frequency, and it was then transferred downward in the image by slight fluidic flow. In contrast, the other green cells remained in the original position. The results indicated that the repulsive force of n-DEP from the strong electric field region acts on the cell in the 1–G well that comprises both band electrodes switched in the n-DEP frequency region. It is noted that p-DEP still acted on cells in wells comprising band electrodes that applied an AC signal in the p-DEP and n-DEP frequency regions, respectively. Thus, this system would make it possible to retrieve target cells selectively from the array of cells and recover them in an outlet without the microdispensers. Figure 1 <p></p>

We provide a novel method to discriminate the cells binding with molecules immobilized on a glass substrate by using the repulsive force of negative dielectrophoresis. The device consists of a lower substrate with interdigitated (IDA) patterns (12 μm in width and 50 μm in gap) of indium-tin oxide (ITO) and an upper ITO substrate disposed on the lower substrate. When an AC voltage is applied to a pair of microband electrodes comprising IDA to the upper ITO substrate connected to the ground, the cells are pressed onto gaps between the microband electrodes, and adhesion of the cells to the substrate is promoted (1st patterning in Fig. A). Subsequently, when an AC voltage is microband electrode “a” against the microband “b” and the upper ITO connected to the ground, the weakest region of the electric field is formed just above the microband “b”, and the cells move there by the repulsive force of negative dielectrophoresis (2nd patterning in Fig. B). At this time, if the cells adhered to the substrate via binding to molecules immobilized on the lower substrate, the cells remain on the gaps. Cells that do not interact with the molecules are expelled on the microband electrode. These procedures provide a rapid method for evaluating the expression of surface proteins that interact with molecules on the lower substrate in as little as 2 min; 60 sec in an adhesion and 60 sec in an expelling. The proposed method was applied to identify the differentiation status of HL60. Undifferentiated HL60 is known to express CD13 but not CD11b, while HL60 differentiated into neutrophils and monocytic lineages is known to up-regulate CD11b expression and down-regulate CD13 expression. In the device pretreated with anti-CD11b antibody, almost all HL60 formed well-defined linear patterns on the gaps between the microband electrodes at the 1st patterning by applying the ac voltage (100 kHz in frequency and 20 Vpp in voltage) for 60 seconds and aligned on the microband electrode “b” after 2nd patterning (day 0 in Fig. B). This result suggests that CD11b rarely expressed in HL60 at the state of undifferentiation. It was also confirmed that the expression levels of CD11b were extremely low by immunobiological fluorescent imaging. Conversely, HL60s differentiated with 1 μM all-trans retinoic acid (ATRA), which is well known as a inducer of HL60 to neutrophil, were randomly adhered on the gaps, and only a few of the adhered HL60s moved onto the microband electrodes after 2nd patterning (day 4 inf Fig. B). The increase of number of cells remaining on the gaps in differentiated HL60 represented the upregulation of CD11b expression. The expression of CD11b in differentiated cells cultured in ATRA-containing medium for 4 days was confirmed by immunostaining. There was a good correlation between the expression levels of CD11b obtained by the present method and those obtained by conventional fluorescent method. In addition, the expression of CD13 was investigated using the device pretreated with anti-CD13. In this case, the majority of undifferentiated cells adhered on the gap between the microband electrode and the differentiated cells were expelled from the gap onto the microband electrode “b”. Thus, using the repulsive force of n-DEP to expel cells unbound to the substrate, the differentiation status can be elucidated in as little as 120 seconds. Figure 1 <p></p>

Introduction: We have developed manipulation techniques to form cell-based arrays by positive dielectrophoresis (p-DEP) and to retrieve target cells from cell−based arrays selectively by negative dielectrophoresis (n-DEP). The novel devices with microwell arrays on microband electrodes were employed to manipulate cells. Hybridomas with the secretion ability of antibodies were trapped to form cell-based arrays. Then, the ability was discriminated by trapping the antibodies to the antigen immobilized on the bottom of wells. Finally, individual cells trapped in microwells were selectively retrieved by regulating the generation of electric fields in individual microwells. The development of series of these techniques could be useful to recover hybridomas producing antibodies with high affinity in large populations of cells without repeated steps of a culture and a limiting serial-dilution. Experimental: The device comprises the upper substrate with indium-tin-oxide (ITO) microband electrodes and the lower substrate with microwell arrays on ITO microband electrodes. Hybridomas producing an anti-rabbit serum albumin (RSA) antibody suspended in the DEP medium were introduced in the channel. AC signal (3 MHz, 2 Vpp) was then applied to the upper and lower microband electrodes with opposite phase to form a cell-based array. Anti-RSA antibodies secreted from hybridomas trapped in the microwells were captured by RSA immobilized on the electrodes. Cell arrays were then treated with anti-mouse IgG antibody conjugated with Alexa 488 (a secondary antibody). Thereafter, a repulsive force of negative dielectrophoresis (n-DEP) was employed to retrieve the target hybridomas from the microwell array. To retrieve the target hybridoma, an AC signal in the n-DEP frequency region was applied to a pair of microband electrodes above and below the microwell with the target hybridoma. Results and discussion: Cell−based arrays were formed with the occupancy efficiency of over 90% in a few seconds by p-DEP. Antibodies secreted in microwell arrays were captured to discriminate target hybridomas in a few hours without repeated steps of a culture and a limiting serial-dilution. Furthermore, hybridomas trapped in microwells were retrieved from the cell−based array by applying an AC signal to band electrodes. The sequential system for forming cell−based arrays, discriminating hybridomas secreting specific antibodies, and retrieving target hybridomas was developed by using the novel microwell array device comprising 3-D microband array electrodes with an orthogonal arrangement.