**4. Construction of neural network by applying soft lithography**

A man-made neural network on electrode can be applied to do basic research of neuroscience, be a kind of biosensor for drug discovery [9] and even be implanted into brain to establish artificial connections that could form the basis of a neural prosthesis[10]. These fields have caused much attention in the world [11]. In the past, there are three strategies to realize a simplified neural network in vitro, such as mechanical fixation-applying spatial restrictions[12,13], physical modification of surface roughness and surface topography[14,15], chemical polymer microfabrication on surface—using soft lithography. Soft lithography is created by Whitesides in 1993[16]. It is used to create chemical structures on surfaces, including µCP, µFN and other downstream techniques. These microfabrication techniques that control both the size and shape of the cell anchored to a particular surface are extremely useful for understanding the inuence of the cell–material interface on the behavior of cells [17, 18].The adhesion and survival of neural cells should be considered firstly for the patterned neural cell culture in vitro. So the selection of appropriate cellattracting substances is an important step for pattern design in micro-contact printing. Ideal substances encourage good cell–substrate interactions, constantly stimulate the cells by substrate-bound chemical, biological, electrical or mechanical signals [19, 20] and even regulate neuritis growth on designed patterns. The most commonly used coating reagents to promote cell adhesion are extracellular matrix (ECM) proteins like laminin (LN), positively charged polymers such as poly-l-lysine (PLL) and synthetic amide-linkage-free compounds such as polyethylenimine (PEI). Therefore, the characteristics of three different substrates, PEI, PLL, LN were compared by the primary neuron culture in our previous work. The PEI characterized with strong positive surface charges was validated to fabricate more continuous and integrated micro-contact printing neural patterns under serum-free culture conditions than PLL, LN[21].

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 microscope.
