Importance of Spatial Arrangement of Cardiomyocyte Network for Precise and Stable On-Chip Predictive Cardiotoxicity Measurement.

One of the advantages of human stem cell-derived cell-based preclinical screening is the reduction of the false negative/positive misjudgment of lead compounds for predicting their effectiveness and risks during the early stage of development. However, as the community effect of cells was neglected in the conventional single cell-based in vitro screening, the potential difference in results caused by the cell number and their spatial arrangement differences has not yet been sufficiently evaluated. Here, we have investigated the effect of the community size and spatial arrangement difference for cardiomyocyte network response against the proarrhythmic compounds from the viewpoint of in vitro cardiotoxicity. Using three different typical types of cell networks of cardiomyocytes, small cluster, large square sheet, and large closed-loop sheet were formed in shaped agarose microchambers fabricated on a multielectrode array chip simultaneously, and their responses were compared against the proarrhythmic compound, E-4031. The interspike intervals (ISIs) in large square sheets and closed-loop sheets were durable and maintained stable against E-4031 even at a high dose of 100 nM. In contrast, those in the small cluster, which fluctuated even without E-4031, acquired stable beating reflecting the antiarrhythmic efficacy of E-4031 from a 10 nM medium dose administration. The repolarization index, field potential duration (FPD), was prolonged in closed-loop sheets with 10 nM E-4031, even though small clusters and large sheets remained normal at this concentration. Moreover, FPDs of large sheets were the most durable against E-4031 among the three geometries of cardiomyocyte networks. The results showed the apparent spatial arrangement dependence on the stability of their interspike intervals, and FPD prolongation, indicating the importance of the geometry control of cell networks for representing the appropriate response of cardiomyocytes against the adequate amount of compounds for in vitro ion channel measurement.

Membrane backtracking at the maximum capacity of nondigestible antigen phagocytosis in macrophages.

The zipper model has been dominantly used to describe the driving mechanism of the engulfment process and its specific identification of antigens during phagocytosis in macrophages. However, the abilities and limitations of the zipper model, capturing the process as an irreversible reaction, have not been examined yet under the critical conditions of engulfment capacity. Here, we demonstrated the phagocytic behavior of macrophages after reaching the maximum engulfment capacity by tracking the progression of their membrane extension during engulfment using IgG-coated nondigestible polystyrene beads and glass microneedles. The results showed that, after macrophages reached their maximum engulfment capacity, they induced membrane backtracking (the reverse phenomenon of engulfment) in both polystyrene beads and glass microneedles, regardless of the difference in the shape of these antigens. We evaluated the correlation of engulfment in simultaneous stimulations of two IgG-coated microneedles and found that each microneedle was regurgitated by the macrophage independently of the advancement or backtracking of membranes on the other microneedle. Moreover, assessing the total engulfment capacity determined by the maximum amount the macrophage was capable of engulfing when imposing each antigen geometry showed that the capacity increased as the attached antigen areas increased. These results indicate that the mechanism of engulfment should imply the following: 1) macrophages have a backtracking function to recover their phagocytic activity after reaching maximal engulfment limit, 2) both phagocytosis and backtracking are local phenomena of the macrophage membrane that operates independently, and 3) the maximum engulfment capacity is determined not only by mere local cell membrane capacity but also by the whole-cell volume increase during simultaneous phagocytosis of multiple antigens by the single macrophages. Thus, the phagocytic activity may entail a hidden backtracking function, adding to the conventionally known irreversible zipper-like ligand-receptor binding mechanism during membrane progression to recover the macrophages that are saturated from engulfing targets beyond their capacity.

Dominant geometrical factors of collective cell migration in flexible 3D gelatin tube structures.

Collective cell migration is a dynamic and interactive behavior of cell cohorts essential for diverse physiological developments in living organisms. Recent studies have revealed the importance of three-dimensional (3D) topographical confinements to regulate the migration modes of cell cohorts in tubular confinement. However, conventional in vitro assays fail to observe cells' behavior in response to 3D structural changes, which is necessary for examining the geometric regulation factors of collective migration. Here, we introduce a newly developed assay for fabricating flexible 3D structures of capillary microtunnels to examine the behavior of vascular endothelial cells (ECs) as they progress through the successive transition across wide or narrow tube structures. The microtunnels with altered diameters were formed inside gelatin-gel blocks by photo-thermal etching with micrometer-sized spot heating of the focused infrared laser absorption. The ECs migrated and spread two-dimensionally on the inner surface of gelatin capillary microtunnels as a monolayer instead of filling the entire capillary. In the straight cylindrical topographical constraint, leading ECs exhibited no apparent diameter dependence for the maximum peak migration velocity. However, widening the diameter in the narrow-wide structures caused a decrease in migration velocity following in direct proportion to the diameter increase ratio, whereas narrowing the diameter in wide-narrow microtunnels increased the speed without obvious correlation between velocity change and diameter change. The results demonstrated the ability of the newly developed flexible 3D gelatin tube structures for collective cell migration, and the findings provide insights into the dominant geometric factor of the emerging migratory modes for endothelial migration as asymmetric fluid flow-like behavior in the borderless cylindrical cell sheets.

In Situ Agarose Microfabrication Technology Using Joule Heating of Micro Ionic Current for On-Chip Cell Network Analysis.

Agarose microfabrication technology is one of the micropatterning techniques of cells having advantages of simple and flexible real-time fabrication of three-dimensional confinement microstructures even during cell cultivation. However, the conventional photothermal etching procedure of focused infrared laser on thin agarose layer has several limitations, such as the undesired sudden change of etched width caused by the local change of absorbance of the bottom surface of cultivation plate, especially on the indium-tin-oxide (ITO) wiring on the multi-electrode array (MEA) cultivation chip. To overcome these limitations, we have developed a new agarose etching method exploiting the Joule heating of focused micro ionic current at the tip of the micrometer-sized capillary tube. When 75 V, 1 kHz AC voltage was applied to the tapered microcapillary tube, in which 1 M sodium ion buffer was filled, the formed micro ionic current at the open end of the microcapillary tube melted the thin agarose layer and formed stable 5 µm width microstructures regardless the ITO wiring, and the width was controlled by the change of applied voltage squared. We also found the importance of the higher frequency of applied AC voltage to form the stable microstructures and also minimize the fluctuation of melted width. The results indicate that the focused micro ionic current can create stable local spot heating in the medium buffer as the Joule heating of local ionic current and can perform the same quality of microfabrication as the focused infrared laser absorption procedure with a simple set-up of the system and several advantages.

Photothermal Agarose Microfabrication Technology for Collective Cell Migration Analysis.

Agarose photothermal microfabrication technology is one of the micropatterning techniques that has the advantage of simple and flexible real-time fabrication even during the cultivation of cells. To examine the ability and limitation of the agarose microstructures, we investigated the collective epithelial cell migration behavior in two-dimensional agarose confined structures. Agarose microchannels from 10 to 211 micrometer width were fabricated with a spot heating of a focused 1480 nm wavelength infrared laser to the thin agarose layer coated on the cultivation dish after the cells occupied the reservoir. The collective cell migration velocity maintained constant regardless of their extension distance, whereas the width dependency of those velocities was maximized around 30 micrometer width and decreased both in the narrower and wider microchannels. The single-cell tracking revealed that the decrease of velocity in the narrower width was caused by the apparent increase of aspect ratio of cell shape (up to 8.9). In contrast, the decrease in the wider channels was mainly caused by the increase of the random walk-like behavior of component cells. The results confirmed the advantages of this method: (1) flexible fabrication without any pre-designing, (2) modification even during cultivation, and (3) the cells were confined in the agarose geometry.

Stepwise neuronal network pattern formation in agarose gel during cultivation using non-destructive microneedle photothermal microfabrication.

Conventional neuronal network pattern formation techniques cannot control the arrangement of axons and dendrites because network structures must be fixed before neurite differentiation. To overcome this limitation, we developed a non-destructive stepwise microfabrication technique that can be used to alter microchannels within agarose to guide neurites during elongation. Micropatterns were formed in thin agarose layer coating of a cultivation dish using the tip of a 0.7 [Formula: see text]-diameter platinum-coated glass microneedle heated by a focused 1064-nm wavelength infrared laser, which has no absorbance of water. As the size of the heat source was 0.7 [Formula: see text], which is smaller than the laser wavelength, the temperature fell to 45 [Formula: see text] within a distance of 7.0 [Formula: see text] from the edge of the etched agarose microchannel. We exploited the fast temperature decay property to guide cell-to-cell connection during neuronal network cultivation. The first neurite of a hippocampal cell from a microchamber was guided to a microchannel leading to the target neuron with stepwise etching of the micrometer resolution microchannel in the agarose layer, and the elongated neurites were not damaged by the heat of etching. The results indicate the potential of this new technique for fully direction-controlled on-chip neuronal network studies.

On-Chip Multiple Particle Velocity and Size Measurement Using Single-Shot Two-Wavelength Differential Image Analysis.

Precise and quick measurement of samples' flow velocities is essential for cell sorting timing control and reconstruction of acquired image-analyzed data. We developed a simple technique for the single-shot measurement of flow velocities of particles simultaneously in a microfluidic pathway. The speed was calculated from the difference in the particles' elongation in an acquired image that appeared when two wavelengths of light with different irradiation times were applied. We ran microparticles through an imaging flow cytometer and irradiated two wavelengths of light with different irradiation times simultaneously to those particles. The mixture of the two wavelength transmitted lights was divided into two wavelengths, and the images of the same microparticles for each wavelength were acquired in a single shot. We estimated the velocity from the difference of its elongation divided by the difference of irradiation time by comparing these two images. The distribution of polystyrene beads' velocity was parabolic and highest at the center of the flow channel, consistent with the expected velocity distribution of the laminar flow. Applying the calculated velocity, we also restored the accurate shapes and cross-sectional areas of particles in the images, indicating this simple method for improving of imaging flow cytometry and cell sorter for diagnostic screening of circulating tumor cells.