Ikurou Suzuki

Non-destructive on-chip cell sorting system with real-time microscopic image processing

Studying cell functions for cellomics studies often requires the use of purified individual cells from mixtures of various kinds of cells. We have developed a new non-destructive on-chip cell sorting system for single cell based cultivation, by exploiting the advantage of microfluidics and electrostatic force. The system consists of the following two parts: a cell sorting chip made of poly-dimethylsiloxane (PDMS) on a 0.2-mm-thick glass slide, and an image analysis system with a phase-contrast/fluorescence microscope. The unique features of our system include (i) identification of a target from sample cells is achieved by comparison of the 0.2-μm-resolution phase-contrast and fluorescence images of cells in the microchannel every 1/30 s; (ii) non-destructive sorting of target cells in a laminar flow by application of electrostatic repulsion force for removing unrequited cells from the one laminar flow to the other; (iii) the use of agar gel for electrodes in order to minimize the effect on cells by electrochemical reactions of electrodes, and (iv) pre-filter, which was fabricated within the channel for removal of dust contained in a sample solution from tissue extracts. The sorting chip is capable of continuous operation and we have purified more than ten thousand cells for cultivation without damaging them. Our design has proved to be very efficient and suitable for the routine use in cell purification experiments.

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.

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.

Constructive formation and connection of aligned micropatterned neural networks by stepwise photothermal etching during cultivation

To understand the topologically dependence of neural network function and its community effects, a constructive approach to forming a model culture system in which we can fully control the spatiotemporal pattern modification during cultivation is useful. We thus newly developed an on-chip multi-electrode array (MEA) system combined with an agarose microchamber (AMC) array to record the firing at multiple cells simultaneously over a long term and to topographically control the cell positions and their connections in order to form two linearly aligned micropatterned networks using additional photothermal etching during cultivation. The electrical connection through the additional neurite connection between two networks in both synchronized spontaneous firings and evoked responses to electrical stimulation was measured, and the localized synaptogenesis at the additional connection point and the propagation by chemical synapses were confirmed. The results show the advantages of AMC/MEA cultivation and measurement methods and indicate they will be useful for investigating community effects by pattern modification during cultivation.

On-Chip Multichannel Action Potential Recording System for Electrical Measurement of Single Neurites of Neuronal Network

We have developed a multielectrode array recording system for single-neurite-firing measurement using an artificially constructed neuronal network on a chip, which has a 10 µm diameter array with electrodes spaced at 50 µm, for noninvasive 64-channel 100 kHz multirecording and the stimulation of a plurality of neurites extending from a single neuron. To improve the signal/noise ratio, the ground plane was set on the multi-electrode-array plane and platinum black was set on each of the 10 µm electrodes. Using this system, we performed a multisite recording of neurites of a single neuron of a rat hippocampal network in cases of both spontaneous firing and evoked responses to electrical stimulations, and estimated the velocity of action potential propagation among neurites of a single neuron from six recording sites. This demonstrated the potential use of our low-noise chip and our high-speed measurement system for the analysis of neuronal network activities at the single-neuron level.

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.