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 the preliminary step for the construction of 3D culture, distribute 160 microliters of the suspension with a cell concentration of 600 to 700 cells per microliter onto the surface of the microbeads monolayer positioned inside the multiwell plates. Then, place the multiwell plates in the incubator with 5% humidified carbon dioxide atmosphere at 37 degrees Celsius.

Six to eight hours after plating, transfer 30 to 40 microliters of the suspension with microbeads and neurons to the area of the MEA delimited by the PDMS constraint. After each transfer, wait about half a minute to allow the microbeads to self-assemble in a hexagonal compact structure. Once all the layers are deposited and spontaneously assembled, add 300 microliters of medium on the top of the area delimited by the PDMS constraint.

Put the 3D structure coupled to the MEA in the incubator at 37 degrees Celsius and 5% carbon dioxide for 48 hours.