**3.4 MEA with microchannels for patterning**

22 Advances in Unconventional Lithography

The mixture of PDMS prepolymer and curing agent was spin-coated on the mold for 40 s at 4000 rpm. The coated mold was cured for 2 hours at 110◦C in a convection oven. The fullycured PDMS-coated mold was soaked in an acetone ultra-sonication bath until the PDMS layer released from the mold. The detached microstencil was rinsed with IPA and DI water. The upside and downside of PDMS microstencil with microholes and microchannels SEM

(a) (b)

Fig. 5. SEM images of PDMS microstencil mold

images was shown in figure 6.

Fig. 6. SEM images of PDMS microstencil

Planar MEA are developed to study electrogenic tissues such as dissociated neuronal cultures (Hiroaki Oka,et al. 1999). They have been widely used with dissociated cultures for a variety of neuroscience investigation including learning and memory and cell-based biosensors for the detection of neurotoxins (Conrad D. James, et al. 2004). But the neurons grow disorderly and cannot form a network so that the function is not the same as the cells in vitro. Combining the patterning technology and MEA forming neuronal networks is the efficient method to research the neurobiology.

μCP, microfluidic patterning technique and microstencil are difficult to operate because the space is too small and the PDMS stamp or stencil can hardly align with MEA. The best and easiest way to forming neuronal networks is to fabricate the microchannels on MEA with polyimide (PI) guiding the cell growing.

MEA were fabricated using a conventional semiconductor process (Guangxin Xiang, et al. 2007). After cleaning the polished quartz glass wafer, the conductive layer of Au/Ti lm (Au 3000 Ǻ and Ti 700 Ǻ) was sputtered. 8 × 8 electrode arrays were left with the photomask protection by standard photolithography. Then, a combination of SiO2/Si3N4/SiO2 (3000 Ǻ /4000 Ǻ /3000 Ǻ) passivation layers was deposited onto the substrate using plasma enhanced chemical vapor deposition (PECVD), and the insulating layers on the electrodes and the bonding-pads were removed by inductively coupled plasma (ICP) (see Fig. 7a). Finally, Negative photosensitive polyimide (AP2210B, Fujifilm Electronic Materials Inc) was spin-coated to form microchannels having a thickness of 3~4μm and photo-etched by the standard procedure to expose the microelectrodes and the terminals (see Fig. 7b).

Fig. 7. Microscopy of the MEA with PI microchannels

GABAergic neurons in the striatum and PC12 cells were cultured on MEA with PI microchannels which were coated with poly-l-lysine (PLL) to promote cell adhesion, (see Fig. 8a, 8b). PI microchannels could be seen between the electrodes and the neural cell can grow along the microchannels. However the nerve cell synapse could not formed along the microchannels. Because the depth of microchannels could not match the neurons and the

Application of Soft Lithography and Micro-Fabrication on Neurobiology 25

For the functional neural network construction on MEA, an inevitable question that should be addressed finally is how to realize accurate opposite between neurons and the electrode under neurons. We assume firstly microfluidic technique may have more advantages than μCP. In subsequent research, we achieved satisfied patterns by microfluidic technique for further research with the help of the progress on parameters of template. Specific neural network were constructed by applying advanced soft lithography above to do the primary cell culture, such as dopaminergic neurons in the substantial nigra and GABAergic neurons in the striatum. The conditions of neuronal adhesion on different patterns (grids and lines) were also observed using several techniques, including atomic force microscopy, immunohistochemistry, transmission electron microscope and scanning electron

In previous study, we examined the ability of another positively charged polymer, polyethyleneimine (PEI), to promote neuronal adhesion, growth and the formation of a functional neuronal network in vitro. PEI, PLL and LN were used to produce grid-shape patterns on glass coverslips by μCP. Post-mitotic neurons from the rat fetal hippocampus were cultured on the different polymers and the viability and morphology of these neurons

The number of cells that adhere to the different substrates after 24 h in culture is shown in Fig. 9. The adhesive effects were evaluated by calculating the ratio of cell numbers that adhere to the grid-like patterns divided by the total area of printed polymer. We found that the positively charged polymers (PLL and PEI) had a signicantly higher level of cell

Fig. 9. The adhesive effects were test by analysis of the number of neurons on the area (mm2) of LN, PEI and PLL grid patterns after 24 h in culture. The asterisks indicated neurons on PEI and PLL patterns had signicantly higher lever than on LN patterns, n = 12,

**4.1 Neural network with rat fetal hippocampal cells by μCP patterns** 

under serum-free culture conditions were observed

microscope.

**4.1.1 Cells adhesion** 

p < 0.05.

attachment than LN (p < 0.05)[13].

PLL could not be guarantee to coat the microchannels effectively after days. There is still a lot of work to do to construct the neuronal networks on MEA to study the cells function as in vitro.

Fig. 8. Microscopy of the cells cultured on MEA with PI microchannels (a) GABAergic neurons in the striatum cultured at 3 days (b) PC 12 cells cultured at 6 days
