- Open Access
Modification of a neuronal network direction using stepwise photo-thermal etching of an agarose architecture
© Suzuki et al; licensee BioMed Central Ltd. 2004
- Received: 11 December 2003
- Accepted: 01 July 2004
- Published: 01 July 2004
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.
- Hippocampal Cell
- Collagen Layer
- Network Pattern
- Heating Spot
Acquisition of the epigenetic information is becoming more and more important for understanding the adaptation mechanism of living systems. One of the main interests of epigenetic studies in neuroscience is how such information is processed and recorded as plasticity within a network pattern, what might be caused by the change in the network pattern or by the degree of complexity related to the network size. One of the best approaches to understanding the meaning of the network pattern and size is to analyze the function of an artificially constructed neural cell network under fully controlled conditions. For many years the formation of neural networks (grown from individual neurons) and the firing patterns of neurons were investigated using microprinting techniques and the fabrication of cultivation substrates [1–3], patterning on silicon-oxide substrates  and three-dimensional structures made using photolithography . Conventional microfabrication techniques provide structures with fine spatial resolution, but are not very effective in studying epigenetic information. Making flexible microstructures with simple steps or changing their shape during cultivation is nearly impossible with conventional techniques since the shape is usually unpredictable and only defined during cultivation.
We have developed a new on-chip cultivation system capable of cultivating cells in a controlled environment using agarose microstructures and a photo-thermal etching method [6, 7]. We can produce microstructures within the agarose layer on the chip using photo-thermal etching i.e. by melting a portion of the agarose layer at the spot of a focused infrared laser beam. This method can be applied prior or during cultivation. We can therefore change the network pattern of nerve cells in real time during cultivation by adding microchannels connecting different microchambers in a step-by-step fashion. This has helped us to understand the meaning of the spatial pattern of a neuronal network by comparing the changes in cell signaling before and after changing the network shape. However, until recently we were not able to control the direction of synaptic connections. We have developed a method to fully control the direction of neural networks within the agarose microchamber (AMCA) system. Our technique can be used to obtain long-term electronic properties of topographically controlled neuronal networks with precise fixation of cell positions and flexible network pattern rearrangement through photo-thermal etching of the agarose layer. This manuscript describes our method for controlling the direction of synaptic connections with the newly developed neural-cell cultivation chip.
On-chip AMCA cell cultivation system
Neural network direction control of hippocampal cells by stepwise photo-thermal etching
Possible damage to collagen layer in agarose microchambers by photo-thermal etching
We have also checked the suitability of a collagen layer for cell cultivation using an AMCA cultivation chip. In the AMCA chip, the collagen layer is between ITO layer and top agarose layer. To manufacture the cultivation wells, we used 1064-nm 35-mW focused laser for 2 s with ×10 objective lens to form 50-μm wide round microchambers. Figure 6(e) is the micrograph taken after 20-hour of cultivating hippocampal cells in these microchambers. Figure 6(f) is the micrograph of the hippocampal cells grown under the same conditions as Figure 6(e), except for the AMCA chip re-coated with collagen after photo-thermal etching. As seen on Figures 6(e) and 6(f) little or no difference was observed between cells grown in different chambers. However, the neuritis on the collagen re-coated chip climbed over the microchambers (Figure 6(f). These results indicate that the collagen layer between ITO layer and agarose layer kept their performance even after photo-thermal etching.
As described above, we can fully control the direction of elongation of neurites by the stepwise photo-thermal etching method. This task is impossible for the conventional pattern control method like microprinting and microstructures. Because neuronal cells have the tendency to elongate one neurite, followed by multiples of short dendrites, the ability to control the elongation direction of the first allows to fully control the direction of neural network. We have therefore created only one tunnel for each AMCA well to guide each neurite into the tunnel in the desired direction. Only after the neurites have sufficiently grown into the tunnels, have we connected the tunnels to other AMCA wells.
Our system uses a 1064-nm focused infrared laser beam, as in [6–9]. This wavelength is not absorbed by water, cells or agar. Only ITO layer is capable of absorbing this wavelength and therefore only a portion of the agar near the ITO layer is melted. This has ensured minimal damage to cultured cells.
We have developed a novel method for controlling the direction of neurite elongation by the stepwise photo-thermal etching. Our on-chip AMCA cell cultivation system combined with a 1064-nm photo-thermal etching method makes it possible to easily and quickly form desired structures within agar layers. We demonstrated that that neural cells can be grown and neural network with the desired direction of neural connections can be created in the AMCA chip. Possible damage of the collagen layer inside the AMCA chip was also investigated to confirm that no distinguishable damage was observed for neural cells cultivation even after the photo-thermal etching procedure. Our system has potential for use in the biological/medical fields for cultivating individual-cell-based networks and measuring their properties.
AMCA cell cultivation chip
AMCA chips were kept at in a constant temperature and under controlled atmosphere and humidity (37°C, 5%, respectively) Aphase-contrast/fluorescent optical microscope (IX-70; with a phase-contrast objective lens, ×20, Olympus, Tokyo, Japan) with a focused 1064-nm infrared laser irradiation unit (max. 1 W; PYL-1-1064-M, IPG Photonics, Oxford, MA, USA) was used to melt the agar layer on the chip. The objective lens in the microscope was used to simultaneously observe the chip surface and to focus the 1064-nm laser. A series of phase-contrast images of cell growth and network formation was acquired by using a charge-coupled device (CCD) camera (CS230, Olympus) and recorded in the computer system with a video capture board.
To attach the collagen onto the ITO surface, the chips were washed twice with 80% ethanol and with PBS, air dried and treated with 2 ml of 150-μg/ml collagen solution (pH 3.0) (Collagen type I-C (from pig skin): Nitta Gelatin, Tokyo, Japan). Following 24 h incubation at room temperature, the chip was washed with PBS once and incubated with 2 ml of a 100-μg/ml Poly-D-Lysine solution (Poly-D-Lysine: SIGMA) for 24 h at room temperature. Following the incubation, the chip was washed with PBS. Collagen and Poly-D-Lysine treated surfaces were coated with 2% (w/v) agarose (ISC BioExpress, GenePure LowMelt: melting temp. 65°C) using a spin-coater (500 rpm for 5 s followe by 4000 rpm for 20 s). The agar-coated chips were placed in a refrigerator at 4°C. The microstructures within the layer were designed using a photo-thermal etching procedure.
Hippocampal cell cultivation
Rat hippocampal cells were obtained from 18-day-old fetuses (E18) following a dissection protocol as described previously . The isolated tissue was incubated in 0.25% trypsin (Sigma) in Ca2+- and Mg2+-free Hank's balanced salt solution (HBSS, Gibco) for 8 min at 37°C. After trypsination the tissue was rinsed in a 2-ml plating medium (Neurobasal medium with B27 supplement, Gibco) five times for 5 min and mechanically dissociated with a fire-polished pipette into single cells. The cells were placed one by one into each agar microchamber with a micropipette and incubated at 37°C with 5% CO2 at saturated humidity (Figure 3). We used a conditioned serum-free medium (Neurobasal medium with 0.074 mg/ml L-glutamine, 50 μg/ml gentamycin, 2% (v/v) B-27 supplement 25 μg/ml, Gibco and 50 μg/ml L-glutamic acid hydrochloride, Sigma). Hippocampal glial cells were cultivated for 2 weeks.
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