Pleurodesis using OK-432 for persistent pleural effusion after cardiac surgery in the neonatal period or early infancy.

OBJECTIVE: To evaluate the efficacy of pleurodesis using OK-432 after cardiac surgery in the neonatal period or early infancy. METHODS: We retrospectively reviewed the data of 11 consecutive patients who underwent cardiac surgery in the neonatal period or early infancy and pleurodesis using OK-432 for persistent postoperative pleural effusion in two institutions. RESULTS: The median age at surgery was 8 days (interquartile range [IR], 2-18) with a body weight of 2.84 kg (IR, 2.30-3.07). The maximum amount of pleural drainage before pleurodesis was 94.7 (IR, 60.2-107.7) ml/kg/day. Pleurodesis was initiated at postoperative day 20 (IR, 17-22) and performed in bilateral pleural spaces in seven patients and unilateral in four. The median numbers of injection were 4 (IR, 3-6) times per patient and 3 (IR, 2-3) times per pleural space. In 10 patients, pleural effusion was decreased effectively, and drainage tubes were removed without reaccumulation within 15 (IR, 12-28) days after initial pleurodesis. However, in one patient, with severe lymphedema, pleural effusion was uncontrollable, resulting in death due to sepsis. Adverse events were observed in nine patients; temporal deterioration of lung compliance and arterial blood gas occurred in two, insufficient drainage requiring new chest tube(s) in five, temporal atrial tachyarrhythmia in one, and lymphedema in four. CONCLUSIONS: Pleurodesis using OK-432 is effective and reliable for persistent postoperative pleural effusion in neonates and early infants. Most of the complications, which derived from inflammatory reactions, were temporary and controllable. However, severe lymphedema is difficult to control.

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.

On-Chip Free-Flow Measurement Revealed Possible Depletion of Macrophages by Indigestible PM2.5 within a Few Hours by the Fastest Intervals of Serial Phagocytosis.

To understand the influence of indigestible particles like particulate matter 2.5 (PM2.5) on macrophages, we examined the time course of the series phagocytosis of indigestible 2 µm polystyrene spheres (PS). Five kinds of antigens were used as samples for phagocytosis; Zymosan, non-coated 2 µm PS, bovine serum albumin (BSA)-coated PS (BSA-PS), IgG-coated PS (IgG-PS), and IgG-BSA-coated PS (IgG/BSA-PS). To keep the surrounding concentration of antigens against single macrophages constant, antigens flowed at a continuous rate of 0.55 µm/s within a culture dish as a free-flow measurement assay (on-chip free-flow method). The interval of series phagocytosis for IgG/BSA-PS was the shortest among five samples; it was six times faster than Zymosan in terms of engulfment frequency, and up to 50 particles were engulfed within two hours, maintaining constant intervals until reaching the maximum number. The rate of increase in the total number of phagocytozed IgG/BSA-PS over time was constant, at 1.5 particles/min, in series phagocytosis with a 33-cell population, indicating that the phagocytosis rate constant remained constant independent of the number of phagocytoses. Reaction model fitting of the results showed that IgG/BSA-PS had the highest efficiency in terms of the phagocytosis rate constant, 2.3 × 10-2 particles/min, whereas those of IgG-PS, BSA-PS, PS, and Zymosan were 1.4 × 10-2, 1.1 × 10-2, 4.2 × 10-3, and 3.6 × 10-3 particles/min, respectively. One-by-one feeding of IgG/BSA-PS with optical tweezers was examined to confirm the phagocytosis intervals, and we found that the intervals remained constant until several times before the maximum number of antigens for engulfment, also indicating no change in the phagocytosis rate constant regardless of the history of former phagocytosis and phagocytosis number. Simultaneous phagocytosis of two IgG-BSA-decorated microneedle engulfments also showed that the initiation and progress of two simultaneous engulfments on the two different places on a cell were independent and had the same elongation velocity. Therefore, each phagocytosis of indigestible antigens does not affect both in series or in simultaneous subsequent phagocytosis until reaching the maximum capacity of the phagocytosis number. The results suggest (1) no change in the phagocytosis rate constant regardless of the history of phagocytosis numbers and attachment timing and positions, and (2) IgG-BSA decoration of indigestible microparticles in blood accelerates their engulfment faster, resulting in a severe shortage of macrophages within the shortest time.

Primary sutureless repair concomitant with the Warden procedure.

A 2-month-old girl who had supracardiac total anomalous pulmonary venous connection (Darling classification type 1b) was referred to our institution. Computed tomography showed that multiple right upper pulmonary veins drained into the vertical vein, near the entry to the superior vena cava. The common pulmonary venous chamber was located lower right than usual, and right upper pulmonary veins were far from the common chamber. We successfully performed primary sutureless repair concomitant with the Warden procedure. Postoperative computed tomography showed unobstructed pulmonary veins and superior vena cava routes, and the vertical vein between right upper and lower pulmonary veins shrank slightly.

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.

Interfacial ferroelectricity in rhombohedral-stacked bilayer transition metal dichalcogenides.

van der Waals materials have greatly expanded our design space of heterostructures by allowing individual layers to be stacked at non-equilibrium configurations, for example via control of the twist angle. Such heterostructures not only combine characteristics of the individual building blocks, but can also exhibit physical properties absent in the parent compounds through interlayer interactions1. Here we report on a new family of nanometre-thick, two-dimensional (2D) ferroelectric semiconductors, where the individual constituents are well-studied non-ferroelectric monolayer transition metal dichalcogenides (TMDs), namely WSe2, MoSe2, WS2 and MoS2. By stacking two identical monolayer TMDs in parallel, we obtain electrically switchable rhombohedral-stacking configurations, with out-of-plane polarization that is flipped by in-plane sliding motion. Fabricating nearly parallel-stacked bilayers enables the visualization of moiré ferroelectric domains as well as electric field-induced domain wall motion with piezoelectric force microscopy. Furthermore, by using a nearby graphene electronic sensor in a ferroelectric field transistor geometry, we quantify the ferroelectric built-in interlayer potential, in good agreement with first-principles calculations. The new semiconducting ferroelectric properties of these four new TMDs opens up the possibility of studying the interplay between ferroelectricity and their rich electric and optical properties2-5.

Content Size-Dependent Alginate Microcapsule Formation Using Centrifugation to Eliminate Empty Microcapsules for On-Chip Imaging Cell Sorter Application.

Alginate microcapsules are one of the attractive non-invasive platforms for handling individual cells and clusters, maintaining their isolation for further applications such as imaging cell sorter and single capsule qPCR. However, the conventional cell encapsulation techniques provide huge numbers of unnecessary empty homogeneous alginate microcapsules, which spend an excessive majority of the machine time on observations and analysis. Here, we developed a simple alginate cell encapsulation method to form content size-dependent alginate microcapsules to eliminate empty microcapsules using microcapillary centrifugation and filtration. Using this method, the formed calcium alginate microcapsules containing the HeLa cells were larger than 20m, and the other empty microcapsules were less than 3m under 4000 rpm centrifugation condition. We collected cell-containing alginate microcapsules by eliminating empty microcapsules from the microcapsule mixture with simple one-step filtration of a 20 m cell strainer. The electrical surface charge density and optical permeability of those cell-encapsulated alginate microcapsules were also evaluated. We found that the surface charge density of cell-encapsulated alginate microbeads is more than double that of cells, indicating that less voltage is required for electrical cell handling with thin alginate gel encapsulation of samples. The permeability of the alginate microcapsule was not improved by changing the reflective index of the medium buffer, such as adding alginate ester. However, the minimized thickness of the alginate gel envelope surrounding cells in the microcapsules did not degrade the detailed shapes of encapsulated cells. Those results confirmed the advantage of alginate encapsulation of cells with the centrifugation method as one of the desirable tools for imaging cell sorting applications.

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.

Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters.

We investigated the dominant rule determining synchronization of beating intervals of cardiomyocytes after the clustering of mouse primary and human embryonic-stem-cell (hES)-derived cardiomyocytes. Cardiomyocyte clusters were formed in concave agarose cultivation chambers and their beating intervals were compared with those of dispersed isolated single cells. Distribution analysis revealed that the clusters' synchronized interbeat intervals (IBIs) were longer than the majority of those of isolated single cells, which is against the conventional faster firing regulation or "overdrive suppression." IBI distribution of the isolated individual cardiomyocytes acquired from the beating clusters also confirmed that the clusters' IBI was longer than those of the majority of constituent cardiomyocytes. In the complementary experiment in which cell clusters were connected together and then separated again, two cardiomyocyte clusters having different IBIs were attached and synchronized to the longer IBIs than those of the two clusters' original IBIs, and recovered to shorter IBIs after their separation. This is not only against overdrive suppression but also mathematical synchronization models, such as the Kuramoto model, in which synchronized beating becomes intermediate between the two clusters' IBIs. These results suggest that emergent slower synchronous beating occurred in homogeneous cardiomyocyte clusters as a community effect of spontaneously beating cells.

Stacking-engineered ferroelectricity in bilayer boron nitride.

2D ferroelectrics with robust polarization down to atomic thicknesses provide building blocks for functional heterostructures. Experimental realization remains challenging because of the requirement of a layered polar crystal. Here, we demonstrate a rational design approach to engineering 2D ferroelectrics from a non-ferroelectric parent compound via employing van der Waals assembly. Parallel-stacked bilayer boron nitride exhibits out-of-plane electric polarization that reverses depending on the stacking order. The polarization switching is probed via the resistance of an adjacently stacked graphene sheet. Twisting the boron nitride sheets by a small angle changes the dynamics of switching thanks to the formation of moiré ferroelectricity with staggered polarization. The ferroelectricity persists to room temperature while keeping the high mobility of graphene, paving the way for potential ultrathin nonvolatile memory applications.

Current-induced switching of proximity-induced ferromagnetic surface states in a topological insulator.

Electrical manipulation of magnetization could be an essential function for energy-efficient spintronics technology. A magnetic topological insulator, possessing a magnetically gapped surface state with spin-polarized electrons, not only exhibits exotic topological phases relevant to the quantum anomalous Hall state but also enables the electrical control of its magnetic state at the surface. Here, we demonstrate efficient current-induced switching of the surface ferromagnetism in hetero-bilayers consisting of the topological insulator (Bi1-xSbx)2Te3 and the ferromagnetic insulator Cr2Ge2Te6, where the proximity-induced ferromagnetic surface states play two roles: efficient charge-to-spin current conversion and emergence of large anomalous Hall effect. The sign reversal of the surface ferromagnetic states with current injection is clearly observed, accompanying the nearly full magnetization reversal in the adjacent insulating Cr2Ge2Te6 layer of an optimal thickness range. The present results may facilitate an electrical control of dissipationless topological-current circuits.

Damus-Kay-Stansel procedure with ventricular septal defect enlargement.

A full-term infant who had tricuspid atresia with transposed great arteries, a ventricular septal defect, subpulmonary stenosis with posterior malalignment of the conus septum, bicuspid pulmonary valve, and a high-takeoff left coronary artery was referred to our institution. The subpulmonary stenosis gradually progressed and cyanosis worsened. We successfully performed a Damus-Kay-Stansel procedure and a bidirectional Glenn shunt concomitant with ventricular septal defect enlargement. The conus septum was resected along with thick fibrous tissue through both semilunar valves (without ventriculotomy). Postoperative echocardiography demonstrated that both the ventricular septal defect and the subpulmonary space were enlarged effectively without semilunar valve regurgitation.

Geometric Understanding of Local Fluctuation Distribution of Conduction Time in Lined-Up Cardiomyocyte Network in Agarose-Microfabrication Multi-Electrode Measurement Assay.

We examined characteristics of the propagation of conduction in width-controlled cardiomyocyte cell networks for understanding the contribution of the geometrical arrangement of cardiomyocytes for their local fluctuation distribution. We tracked a series of extracellular field potentials of linearly lined-up human embryonic stem (ES) cell-derived cardiomyocytes and mouse primary cardiomyocytes with 100 kHz sampling intervals of multi-electrodes signal acquisitions and an agarose microfabrication technology to localize the cardiomyocyte geometries in the lined-up cell networks with 100-300 µm wide agarose microstructures. Conduction time between two neighbor microelectrodes (300 µm) showed Gaussian distribution. However, the distributions maintained their form regardless of its propagation distances up to 1.5 mm, meaning propagation diffusion did not occur. In contrast, when Quinidine was applied, the propagation time distributions were increased as the faster firing regulation simulation predicted. The results indicate the "faster firing regulation" is not sufficient to explain the conservation of the propagation time distribution in cardiomyocyte networks but should be expanded with a kind of community effect of cell networks, such as the lower fluctuation regulation.

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.