Search Results (6)

Axon polarization is a fundamental process in neuronal development, providing the structural basis for directional signaling in neural circuits. Precise control of axon specification is, thus, essential for the bottom-up construction of neuronal networks with defined architecture and connectivity. Although neurite length and elongation timing have both been implicated as determinants of axonal fate, their relative contributions have remained unresolved due to technical limitations in manipulating these factors independently in conventional culture systems. Here, we developed a constructive neuroengineering platform based on modifiable agarose gel microstructures that enables dynamic, in situ control of neurite outgrowth length and timing during neuronal cultivation. This approach allowed us to directly address whether axon polarization depends primarily on neurite length or the order of neurite extension. Using a single-neurite elongation paradigm, we quantitatively defined two length thresholds for axon specification: a critical length of 43.3 μm, corresponding to a 50% probability of axonal differentiation, and a definitive length of 95.4 μm, beyond which axonal fate was reliably established. In experiments involving simultaneous or sequential elongation of two neurites, we observed that neurite length—not elongation order—consistently predicted axonal identity, even when a second neurite was introduced after the first had already begun to grow. The presence of a competing neurite modestly elevated the effective critical length, suggesting inhibitory interactions that modulate length thresholds. These findings provide the first direct experimental confirmation that neurite length is the primary determinant of axon polarization and demonstrate the utility of constructive microfabrication approaches for dissecting fundamental principles of neuronal polarity. Our platform establishes a powerful experimental foundation for future efforts to achieve complete control over axon and dendrite orientation during the engineered construction of functional neuronal circuits. Full article

Constructing stable and flexible neuronal networks with multi-neurite wiring is essential for the in vitro modeling of brain function, connectivity, and neuroplasticity. However, most existing neuroengineering platforms rely on static microfabrication techniques, which limit the ability to dynamically control circuit architecture during cultivation. In this study, we developed a modifiable agarose gel-based platform that enables real-time microstructure fabrication using an infrared (IR) laser system under live-cell conditions. This approach allows for the stepwise construction of directional neurite paths, including sequential microchannel formation, cell chamber fabrication, and controlled neurite–neurite crossings. To support long-term neuronal health and network integrity in agarose microstructures, we incorporated direct glial co-culture into the system. A comparative analysis showed that co-culture significantly enhanced neuronal adhesion, neurite outgrowth, and survival over several weeks. The feeder layer configuration provided localized trophic support while maintaining a clear separation between glial and neuronal populations. Dynamic wiring experiments further confirmed the platform’s precision and compatibility. Neurites extended through newly fabricated channels and crossed pre-existing neurites without morphological damage, even when laser fabrication occurred after initial outgrowth. Time-lapse imaging showed a temporary growth cone stalling at crossing points, followed by successful elongation in all tested samples. Furthermore, the direct laser irradiation of extending neurites during microstructure modification did not visibly impair neurite elongation, suggesting minimal morphological damage under the applied conditions. However, potential effects on molecular signaling and electrophysiological function remain to be evaluated in future studies. Together, these findings establish a powerful, flexible system for constructive neuroengineering. The platform supports long-term culture, real-time modification, and multidirectional wiring, offering new opportunities for studying neural development, synaptic integration, and regeneration in vitro. Full article

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. Full article

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. Full article

Control over spatial distributions and patterns of individual neurons and their neurites provides an essential tool for studying the meaning of neuronal network patterns. Moreover, the complete direction control of synaptic connections between cells in each neuronal network is also essential to investigate detailed information on the relationship between the forward and feedback signaling among the cells. Here, we have developed a method for topographical control of the direction of synaptic connections within a living neuronal network using a new type of individual-cell-based on-chip cell-cultivation system with an agarose microfabrication technology. The advantages of this system include the ability to control positions and number of cultured cells, as well as flexible control of the direction of elongation of axons and dendrites with stepwise melting of a thin agarose layer coated on the cultivation chip with a focused infrared laser beam even during cultivation without any destructive damage on cells. Using this system, we succeeded in forming a fully direction-controlled single-cell-based neuronal network from individual rat hippocampal cells. In this meeting, we discuss the potential damage of heat to cells during stepwise melting of agarose and demonstrate the ability of our on-chip agarose microfabrication method for individual cell-based neural networks. Full article

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. Full article