伊藤 弘明

Revealing the ion distributions on a charged lipid membrane in aqueous solution under the influence of long-range interactions is essential for understanding the origin of the stability of the bilayer...

Microfluidics is a powerful tool to precisely control fluids as well as to manipulate suspended small particles in a micrometer-sized space [...]

Poly(ethylene glycol)-conjugated amphiphilic block copolymers, which contain degradable hydrophobic blocks connected by a rigid hydrogen-bonding motif (RHM), are developed to yield anisotropic nanoassemblies in a manner independent of the crystalline nature of the hydrophobic block. The all-atom and coarse-grained molecular dynamics simulations suggest that the anisotropic alignment of the block copolymers can be attributed to the π-πinteractions of the RHM and the crystallizable hydrophobic blocks help maintain the aligned structure. Light scattering analysis of the polymer assemblies demonstrates the formation of nonspherical assemblies by the RHM-containing block copolymers with an amorphous hydrophobic block; this indicates the strong contribution of the RHM to the directed assembly of the block copolymers, unlike crystallization-driven self-assembly. Enhanced cell proliferation is observed in cell cultures containing normal human fibroblasts in the presence of the anisotropic polymer assemblies with aspect ratios greater than 12 or lengths greater than 310 nm.

Droplet-based microfluidics is a powerful tool for producing monodispersed micrometer-sized droplets with controlled sizes and shapes; thus, it has been widely applied in diverse fields from fundamental science to industries. Toward a simpler method for fabricating microparticles with front–back asymmetry in their shapes, we studied anisotropic gelation of alginate droplets, which occurs inside a flow-focusing microfluidic device. In the proposed method, sodium alginate (NaAlg) aqueous phase fused with a calcium chloride (CaCl2) emulsion dispersed in the organic phase just before the aqueous phase breaks up into the droplets. The fused droplet with a front–back asymmetric shape was generated, and the asymmetric shape was kept after geometrical confinement by a narrow microchannel was removed. The shape of the fused droplet depended on the size of prefused NaAlg aqueous phase and a CaCl2 emulsion, and the front–back asymmetry appeared in the case of the smaller emulsion size. The analysis of the velocity field inside and around the droplet revealed that the stagnation point at the tip of the aqueous phase also played an important role. The proposed mechanism will be potentially applicable as a novel fabrication technique of microparticles with asymmetric shapes.

Enhancers regulate gene expressions in a tissue- and pathology-specific manner by altering its activities. Plasma levels of atrial and brain natriuretic peptides, encoded by the Nppa and Nppb, respectively, and synthesized predominantly in cardiomyocytes, vary depending on the severity of heart failure. We previously identified the noncoding conserved region 9 (CR9) element as a putative Nppb enhancer at 22-kb upstream from the Nppb gene. However, its regulatory mechanism remains unknown. Here, we therefore investigated the mechanism of CR9 activation in cardiomyocytes using different kinds of drugs that induce either cardiac hypertrophy or cardiac failure accompanied by natriuretic peptides upregulation. Chronic treatment of mice with either catecholamines or doxorubicin increased CR9 activity during the progression of cardiac hypertrophy to failure, which is accompanied by proportional increases in Nppb expression. Conversely, for cultured cardiomyocytes, doxorubicin decreased CR9 activity and Nppb expression, while catecholamines increased both. However, exposing cultured cardiomyocytes to mechanical loads, such as mechanical stretch or hydrostatic pressure, upregulate CR9 activity and Nppb expression even in the presence of doxorubicin. Furthermore, the enhancement of CR9 activity and Nppa and Nppb expressions by either catecholamines or mechanical loads can be blunted by suppressing mechanosensing and mechanotransduction pathways, such as muscle LIM protein (MLP) or myosin tension. Finally, the CR9 element showed a more robust and cell-specific response to mechanical loads than the -520-bp BNP promoter. We concluded that the CR9 element is a novel enhancer that responds to mechanical loads by upregulating natriuretic peptides expression in cardiomyocytes.

We investigated the spontaneous deformation and fission of a tetradecane droplet containing palmitic acid (PA) on a stearyltrimethylammonium chloride (STAC) aqueous solution. In this system, the generation and rupture of the gel layer composed of PA and STAC induce the droplet deformation and fission. To investigate the characteristics of the droplet-fission dynamics, we obtained the time series of the number of the droplets produced by fission and confirmed that the number has a peak at a certain STAC concentration. Since the fission of the droplet should be led by the deformation, we analyzed four parameters which may relate to the fission dynamics from the spatiotemporal correlation of the droplet-boundary velocity. We found that the parameter which corresponds to the expansion speed had the strongest positive correlation among them, and thus we concluded that the faster deformation would be the key factor for the fission dynamics.

The system in which a small rigid ball is bouncing repeatedly on a heavy flat table vibrating vertically, so-called the bouncing ball system, has been widely studied. Under the assumption that the table is vibrating with a piecewise polynomial function of time, the bifurcation diagram changes qualitatively depending on the order of the polynomial function. We elucidate the mechanism of the difference in the bifurcation diagrams by focusing on the two-period solution. In addition, we derive the approximate curve of the branch close to the period-doubling bifurcation point in the case of the piecewise cubic function of time for the table vibration. We also performed numerical calculation, and we demonstrate that the approximations well reproduce the numerical results.

In cell culture, the pH of the culture medium is one of the most important conditions. However, the culture medium may have non-uniform pH distribution due to activities of cells and changes in the environment. Although it is possible to measure the pH distribution with an existing pH meter using distributed electrodes, the method involves direct contact with the medium and would greatly increase the risk of contamination. Here in this paper, we propose a computed tomography (CT) scan for measuring pH distribution using the color change of phenol red with a light-emitting diode (LED) light source. Using the principle of CT scan, we can measure pH distribution without contacting culture medium, and thus, decrease the risk of contamination. We have developed the device with a LED, an array of photo receivers and a rotation mechanism. The system is firstly calibrated with different shapes of wooden objects that do not pass light, we succeeded in obtaining their 3D topographies. The system was also used for measuring a culture medium with two different pH values, it was possible to obtain a pH distribution that clearly shows the boundary.

This paper discusses an on-chip Red Blood Cell (RBC) deformability checker with vision analyzer. The system is composed of a PDMS chip including three microfluidic channels, a microscope for appropriately enlarging RBC, and a high-speed vision for observing both the cell size and behaviors in the channel, respectively. We particularly focus on how to analyze the deformability of single cells by the proposed vision analyzer. The analyzer can detect the diameter and velocity of each cell inside and outside the test channel. Based on the information, we can obtain a deformability map which shows the relationship between cell deformation and transit velocity. The results obtained by the proposed vision analyzer are compared to the manual evaluation with a tuning parameter based on gray level control. The comparison results show that the error is less than 5% between vision-based and manual evaluations.

Physical interactions of four major green tea catechin derivatives with cell membrane models were systemically investigated. Catechins with the galloyl moiety caused the aggregation of small unilamellar vesicles and an increase in the surface pressure of lipid monolayers, while those without did not. Differential scanning calorimetry revealed that, in a low concentration regime (≤10 μM), catechin molecules are not significantly incorporated into the hydrophobic core of lipid membranes as substitutional impurities. Partition coefficient measurements revealed that the galloyl moiety of catechin and the cationic quaternary amine of lipids dominate the catechin-membrane interaction, which can be attributed to the combination of electrostatic and cation-π interactions. Finally, we shed light on the mechanical consequence of catechin-membrane interactions using the Fourier-transformation of the membrane fluctuation. Surprisingly, the incubation of cell-sized vesicles with 1 μM galloyl catechins, which is comparable to the level in human blood plasma after green tea consumption, significantly increased the bending stiffness of the membranes by a factor of more than 60, while those without the galloyl moiety had no detectable influence. Atomic force microscopy and circular dichroism spectroscopy suggest that the membrane stiffening is mainly attributed to the adsorption of galloyl catechin aggregates to the membrane surfaces. These results contribute to our understanding of the physical and thus the generic functions of green tea catechins in therapeutics, such as cancer prevention.

Red blood cell responses during a long-standing load were experimentally investigated. With a high-speed camera and a high-speed actuator, we were able to manipulate cells staying inside a microfluidic constriction, and each cell was compressed due to the geometric constraints. During the load inside the constriction, the color of the cells was found to gradually darken, while the cell lengths became shorter and shorter. According to the analysis results of a 5 min load, the average increase of the cell darkness was 60.9 in 8-bit color resolution, and the average shrinkage of the cell length was 15% of the initial length. The same tendency was consistently observed from cell to cell. A correlation between the changes of the color and the length were established based on the experimental results. The changes are believed partially due to the viscoelastic properties of the cells that the cells' configurations change with time for adapting to the confined space inside the constriction.

Large deformability of erythrocytes in microvasculature is a prerequisite to realize smooth circulation. We develop a novel tool for the three-step "Catch-Load-Launch" manipulation of a human erythrocyte based on an ultra-high speed position control by a microfluidic "robotic pump". Quantification of the erythrocyte shape recovery as a function of loading time uncovered the critical time window for the transition between fast and slow recoveries. The comparison with erythrocytes under depletion of adenosine triphosphate revealed that the cytoskeletal remodeling over a whole cell occurs in 3 orders of magnitude longer timescale than the local dissociation-reassociation of a single spectrin node. Finally, we modeled septic conditions by incubating erythrocytes with endotoxin, and found that the exposure to endotoxin results in a significant delay in the characteristic transition time for cytoskeletal remodeling. The high speed manipulation of erythrocytes with a robotic pump technique allows for high throughput mechanical diagnosis of blood-related diseases.

We propose a microscopic system that can perform simultaneous time-lapse recordings for studying cell behavior on different substrates. The microscopic objective lens is controlled up and down so that the focal plane is matched with different heights of each surface. The captured video includes two types of images with a given time interval. Later, the focused image corresponding to each substrate is rearranged every time segment. To confirm the working principle, we show the experimental results for smooth muscle cells (SMCs) by using two different substrates.

A novel microfluidic system for applying cyclic pressure to cells during incubation is developed and tested in this work. The cyclic pressure generates stress stimulus to the cells, and is named "Cell Exercise" in this work. The goal of this paper is to design an "On-Chip Cell Gym" where we can observe what happens during the cell exercise in real-time. We design the cell gym on a PDMS chip composed of two parallel chamber arrays, so that we can directly observe the difference with and without Cell Exercise. Cells in one group of chamber is cultured under cell exercise mode and the other one is under none cell exercise mode. Dramatic growth of cell stress fibers is observed in the group of cell exercise with respect to the group without any exercise.

"Cell Pinball" is a phenomenon whereby rotating red blood cells (RBCs) move like pinballs inside a microfluidic channel where the cell inertia is negligible. The goal of this paper is to discuss the location of the actual rotational axis through the careful comparison between the contour and contact shapes of a moving RBC. By switching the reflection interference contrast (RIC) and the phase contrast (PhC) microscopes with the mean cycle time of 0.440 s, we found that the difference between the trajectories of each centroid of the visualized RBC by RIC and PhC is less than 0.8 mu m.

This paper focuses on the comparison of red blood cell (RBC) deformability under continuous and repetitive loadings. We utilized a feedback position-control system and a narrow microfluidic channel for applying different deformation patterns on RBCs. According to the analyses of shape recoveries with different patterns, we found, for the first time, that the mechanical responses of RBCs upon continuous and repetitive loadings are almost the same within the error among cellular individualities as long as the total duration of the loading is the same. The result indicates that the internal mechanical stress on RBCs accumulates even if the apparent cell shape recovers. The finding provides quantitative insights for the systematic comparison among various exisiting measurement methods of mechanical responses of cells.