**4.1.2 Cells viability**

In Fig.10, We show that neurons cultured on the PEI- and PLL-coated surfaces adhered to and extended neurites along the grid-shape patterns, whereas neurons cultured on the LNcoated coverslips clustered into clumps of cells. In addition, we found that the neurons on the PEI and PLL-coated grids survived for more than 2 weeks in serum-free conditions, whereas most neurons cultured on the LN-coated grids died after 1 week[13].

Fig. 10. Images obtained using the phase contrast microscope, showing cells cultured on PEI, PLL and LN polymeric lms at different time points. (a, d, and g) Representative images of neurons cultured on PEI grid patterns at 24 h, 7 days, and 14 days, respectively, show that neurons adhere and grow accurately along the PEI grids at differently time points. (b, e, and h) Representative images of neurons cultured on PLL grid patterns at 24 h, 7 days, and 14 days, respectively. (c, f, and i) Representative images of neurons cultured on LN grid patterns at 24 h, 7 days, and 14 days, respectively. The images (c and f) show that the neurons on the LNgrid patterns often accumulate at the cross points of the grids. The image (i) shows that most neurons disappear after 14 days in culture and only small areas of the neural cells exist. (a–c) Bar = 100 μm; (d–i) bar = 50 μm.

## **4.2 Specific neural network with two relative neurons, such as dopaminergic neurons and GABAergic neurons**

There is a closely relationship in the respect of function and structure between dopaminergic neurons in the substantial nigra and GABAergic neurons in the striatum. As well-known, Parkinson's disease is due to the loss and injuries of dopaminergic neuron in substantia nigra which cause a decrease in nerve fibers projected to the new striatum. Finally, reasonable synaptic connections and neural network can not be established. Therefore, it is expected to establish cell models to investigate the relationship between these two kinds of coherent neurons and construct an artificial neural network in vitro by the application of soft lithography. In present work, specific neural network with dopaminergic neurons and GABAergic neurons co-culture was established by μCP PEI grid patterns. Meanwhile, PEI was validated again to fabricate more continuous and integrated neural patterns by using μCP and microfluidic technique both.
