Constructing Patterned Neuronal Circuits on a Multi-Electrode Array

First, clean the multielectrode array by washing it under tap water, and then sonicating it in a concentrated enzymatic detergent. Repeat these steps three times. Then, sonicate the MEA in pure distilled water three times. After which, under a hood, wash the MEA with distilled water before sterilizing it with UV light for 30 minutes.

Now, attach a stencil to a prepared micromanipulator. Under an inverted microscope, inspect and align the patterned structure to match the electrodes of the MEA. Then, use the micromanipulator to lower the stencil to the MEA surface. Once attached, raise the micromanipulator. Now, transfer the MEA to the vacuum chamber for 15 minutes. Then, apply a 1-milliliter drop of PDL to the stencil, and return it to the vacuum chamber for 2 cycles of 20 minutes.

Let the PDL dry overnight. The next day, remove the PDMS stencils from the MEAs using tweezers. Lastly, UV-sterilize the MEAs for 7 minutes. They are then ready for the cells. In preparation, culture cells and prepare suspensions as in the text protocol. After resuspending the required number of cells, plate them at the center of the patterned area on the MEA or coverslip, and MEA should be loaded with 100-microliter volumes, and a coverslip accommodates 1,000 microliters. Incubate the plated cells for 40 minutes. Then, add plating medium.

To maintain the cells every 4 days, add back fresh supplemented growth medium. After the sixth or seventh days, neuronal connections between the modules should start forming.