Building Three-Dimensional Neuronal Networks Coupled to Micro-Electrode Arrays

Take a suspension of rat embryo hippocampal neurons.

Transfer the neurons onto the adhesion factor-coated active area of a microelectrode array, or MEA. This active area is confined by a polymer structure.

Incubate the MEA. Neurons adhere to the coated MEA active area, forming a 2D neuronal network.

Additionally, transfer the neurons onto an adhesion factor-coated glass microbead monolayer in membrane inserts within a multi-well plate.

Incubate for the neurons to attach to the adhesion factor-coated beads.

Transfer the microbeads with adherent neurons to the confined MEA area containing the neuronal network.

Microbeads self-assemble to form a hexagonal structure.

Repeat the microbead transfer on the MEA several times, building layers that self-assemble, forming a packed 3D structure.

Add media to the MEA confined active area and incubate.

Neurons grow, forming an interconnected 3D neuronal network.

Plate the cells at a density of 2,000 cells per square millimeter onto the active area of the MEA, defined by the PDMS constraint to create a 2D neuronal network. Next, place the MEA device into the incubator with 5% humidified carbon dioxide atmosphere at 37 degrees Celsius.

To complete