Stepwise pattern modification of neuronal network in photo-thermally-etched agarose architecture on multi-electrode array chip for individual-cell-based electrophysiological measurement

Abstract

We have developed a procedure for stepwise topographical control of network patterns and neurite connection directions between adjacent living neurons using an individual-cell-based on-chip multi-electrode array (MEA) cell cultivation system with an agarose microchamber (AMC) array. This procedure enables flexible and precise control of the cell positions and easy and flexible control of the pattern modification of connections between the cells in AMCs through stepwise photo-thermal etching in which a portion of the agarose layer on the chip is melted with a 1480 nm infrared laser beam even during cultivation. With adequate laser power and this stepwise procedure, we can fabricate narrow micrometer-order grooves (microchannels) during cultivation in a stepwise manner. Using this procedure, we controlled the direction of elongation of axons and dendrites selectively and confirmed the direction by immunostaining. We also demonstrated electrophysiological one-way transmission of signals among aligned hippocampal neurons in which the directions of the neurite connections were controlled using this stepwise photo-thermal etching procedure. These results demonstrate the potential of full direction control of neurite connections between neurons using stepwise photo-thermal etching to form microchannels one by one in an on-chip AMC/MEA cell cultivation system. We can thus better understand the meaning of neuronal network patterns and connection directions.

An automated microdrop delivery system for neuronal network patterning on microelectrode arrays

“…Jimbo et al (1993) first monitored the electrical activity of patterned neuronal networks with MEAs. More recently, Suzuki et al (2004Suzuki et al ( , 2005 demonstrated the possibility of specifically designing a neural architecture on MEA substrates; they were able to control the growth and connections of a hippocampal cell culture by using a laser to dynamical ablate an agarose layer to create channels and pits that entrap neurons and guide neuronal growth. Other examples in the literature are related to the patterning or confinement of neurons extracted from invertebrate (Fromherz, 2003;Claverol-Tinturé et al, 2005, in press).…”

Novel Technology to Assay the Multicellular Network: On-Chip Cellomics Technology

“…In contrast, as a 1480-nm IR laser has absorption to water, all the agar on the light pathway is heated and melted. The changed sol state agar is dispersed into the agar gel and holes or tunnels are formed in the agar layer applied even during cultivation, so we can change the network pattern of nerve cells during cultivation by adding microchannels between two adjacent microchambers in a step-by-step fashion Suzuki et al 2005). We used this new type of noncontact three-dimensional photothermal etching for the agar-microetching exploiting the characteristics of the two different infrared laser beam wavelengths (1480 and 1064 nm).…”

Automatic positioning and sensing microelectrode array (APSMEA) for multi-site electrophysiological recordings

“…Traditionally, two approaches have been used to control the locations of dissociated neurons on a certain region of the substrate, either employing containment such as micro-wells, micro-channels and micro-drops (Jimbo et al, 1993;Macis et al, 2007;Maher et al, 1999;Suzuki et al, 2005;Zeck and Fromherz, 2001), or adopting appropriate materials for surface patterning (Chang et al, 2003;Kleinfeld et al, 1988;Nam et al, 2004;Scholl et al, 2000). Indeed these approaches achieved a lot of successes, but it is still a challenge for precise manipulation of cells or treating the surface regions.…”