Yoshihiro Sugio

Modification of a neuronal network direction using stepwise photo-thermal etching of an agarose architecture.

Control over spatial distribution of individual neurons and the pattern of neural network provides an important tool for studying information processing pathways during neural network formation. Moreover, the knowledge of the direction of synaptic connections between cells in each neural network can provide detailed information on the relationship between the forward and feedback signaling. 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 microchamber array (AMCA). The advantages of this system include the possibility to control positions and number of cultured cells as well as flexible control of the direction of elongation of axons through stepwise melting of narrow grooves. Such micrometer-order microchannels are obtained by photo-thermal etching of agarose where a portion of the gel is melted with a 1064-nm infrared laser beam. Using this system, we created neural network from individual Rat hippocampal cells. We were able to control elongation of individual axons during cultivation (from cells contained within the AMCA) by non-destructive stepwise photo-thermal etching. We have demonstrated the potential of our on-chip AMCA cell cultivation system for the controlled development of individual cell-based neural networks.

An agar-based on-chip neural-cell-cultivation system for stepwise control of network pattern generation during cultivation

We have developed a new type of single-cell based on-chip cell-cultivation system with an agarose microchamber (AMC) array and a photo-thermal etching module for step-by-step topographical control of the network patterns of living neural cells during long-term cultivation. The advantages of this system are that (1) it can control positions and numbers of cells for cultivation by using agar-based microchambers, and (2) it can change the neural network complexity during cultivation by photo-thermal melting a portion of agar at the focal point of a 1064 nm infrared laser beam. This laser wavelength is permeable with respect to water and agarose, and it is only absorbed at the thin chromium layer on the chromium-coated glass slide surface at the bottom of the agarose layer. With adequate laser power, we can easily fabricate narrow tunnel-shaped channels between the microchambers at the bottom of the agar layer without the complicated steps conventional microfabrication processes entail even during cultivation; we demonstrated that rat hippocampal cells in two adjacent chambers formed fiber connections through new connections between chambers after these had been photo-thermally fabricated. We also verified the fiber connection between those cells by using calcium-based fluorescent microscopy. These results indicate that this system can potentially be used for studying the complexity of neural network patterns for epigenetic memorization.

Individual-cell-based electrophysiological measurement of a topographically controlled neuronal network pattern using agarose architecture with a multi-electrode array

We have developed a new type of individual-cell-based electrophysiological measurement method using an on-chip multi-electrode array (MEA) cell-cultivation system with an agarose microchamber (AMC) array for topographical control of the network patterns of a living neuronal network. The advantages of this method are that it allows the recording of the firing of multiple cells simultaneously for weeks without contamination using the MEA, and that it allows control of the cell positions and numbers, and their connections for cultivation using AMCs with microchannels fabricated by photothermal etching where a portion of the agarose layer is melted with a 1480 nm infrared laser beam. Using this method, we formed an individual-cell-based neural network pattern of Rat hippocampal cells within the AMC array without cells escaping from the electrode positions in the microchamber during a thirteen-day cultivation, and could record the cell firing of lined-up hippocampal cells in response to 20 µA, 5 kHz stimulation via an electrode. This demonstrated the potential of our on-chip AMC/MEA cell cultivation method for long-term single-cell-based electrophysiological measurement of a neural network system for understanding the topographical meaning of neuronal network patterns.

Single-cell-based electrophysiological measurement of a topographically controlled neuronal network using agarose architecture with a multi-electrode array

Understanding the indeterminate development of neural networks requires a better understanding of the epigenetic information acquisition processes of neural cells. A main interest in the field of neuroscience is how such information is processed and recorded as plasticity within a network pattern. Also of interest is what might be caused by changes in the network pattern or by changes in the degree of complexity related to network size. One of the best approaches to understanding the meaning of network pattern and size is to analyze the functions of an artificially constructed neural cell network under fully controlled conditions. Neurophysiologists have investigated single-cell-based neural network cultivation and examined the firing pattems of single neurons using cultivation substrates fabricated using microprinting techniques, using patterned silicon-oxide substrates, and using three-dimensional structures fabricated using photolithography. Although these conventional microfabrication techniques provide structures with fine spatial resolution, effective approaches to studying epigenetic information acquisition are still being sought. With conventional techniques, it is still hard to construct flexible microstructures simply or to change their shape during cultivation since the shape is usually unpredictable and only defined during cultivation. We have developed a single-cell-based on-chip multi-electrode array (MEA) cell cultivation system with an agarose microchamber (AMC) array for topographical control of the network pattems of living neurons’”). This system enables flexible and precise control of the cell positions for cultivation using the AMCs, as well as easy and flexible control of the pattern of connections between the AMCs through photo-thermal etchin in which a portion of the agarose layer is melted with a 1480-nm infrared laser grooves (microchannels) during cultivation that can be used to combine neighboring AMCs, enabling comparison of the network response of neural network patterns with different complexities. Using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the AMC array without cells escaping from the electrode positions in the microchamber and recorded the cell firing (Fig.2). Moreover, we have created a single-cell-based neural network with a stepwise change in the network formation and recorded the electrophysiological changes of the neurons’). These results demonstrate the potential for creating the next stage of single-cell-based neuronal networks and measuring their properties and for use in the biological and medical fields.

Stepwise pattern modification of neuronal network during cultivation using photo-thermal etching of agarose architecture

We have developed a new type of on-chip cell cultivation system using an agarose microchamber (AMC) array and a photo-thermal etching method, thus enabling topographical control of neuronal network pattern step-by-step during cell cultivation. By using photo-thermal etching (micro melting) method, the number of microtunnel connecting microchambers can be easily increased, even during cell cultivation, according to the progress of the neuronal network formation. To demonstrate the capability of this system for topographical control of network formation, we cultured hippocampal neurons in this AMC array. We found that the cells in microchambers made fiber connections through microtunnels. Furthermore the cells even made fiber connections through additional microtunnels fabricated during cultivation by photo-thermal etching. The results showed that the photo-thermal etching could be used during cultivation without damaging cells.

On-Chip Neural Cell-Cultivation System for Long-Term Observation with Multi-Electrode and Microchamber Arrays

A new type of multi-electrode array system for long-term neural-cell cultivation has been developed. The core of the system is a 9-room microchamber array, formed of thick SU-8 photoresist, with a 9-channel multi-electrode array connected to each chamber. The biotin-coated microchamber array plate is covered with a streptavidin-decorated cellulose semi-permeable membrane, fixed on the surface of the plate by streptavidin-biotin attachment, thus separating the microchambers from the nutrient medium circulating above. The advantages of this system are as follows: (1) Contamination-free cultivation of neural cells is achieved by sealing the semi-permeable membrane lid on the microchamber array. (2) The simple, flexible design of the microchamber array enables the positions of the cells to be fixed between thick-photoresist walls 1–5 micrometers wide and 25 micrometers high. (3) Optical microscope observation and electrical recording enable long-term simultaneous analysis of the cells. Our experiments showed that nerve cells cultured in the microchamber array were grown safely, without any contamination. These results demonstrate the potential usefulness of our multi-electrode array system.