This article outlines a protocol for preparing multi-electrode arrays (MEAs) for neuronal cell culture. It details the steps for cleaning, sterilizing, and coating the MEAs with a matrix to facilitate neuronal growth and circuit formation.
Take a cleaned and sterilized multi-electrode array, or MEA, a device containing microelectrodes to obtain and deliver neural signals.
Attach a patterned polymeric stencil to a micromanipulator and place it on the MEA surface.
Under a microscope, align the patterned stencil with the MEA's electrodes and attach it to create modules.
Remove the micromanipulator.
Transfer the MEA to a vacuum chamber. Add a matrix solution to the stencil and apply a vacuum to ensure an even matrix coating.
Allow the matrix to dry.
Remove the stencil, and sterilize the MEA by exposing it to ultraviolet light.
Add neuronal cells to the center of the MEA and allow the cells to attach to the matrix layer.
Next, add growth media and incubate.
Nutrients from the media and the extracellular matrix enable the cells to grow and extend projections, forming interconnected patterned neuronal circuits whose signals can be recorded using the MEA.
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.
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