Developing an Engineered Silk-Collagen-Based 3D Model of Polarized Neural Tissue

Place donut-shaped, poly-D-lysine-coated, porous silk scaffolds into a multi-well plate.

Add culture media to hydrate the scaffolds, facilitating cell attachment.

Aspirate excess media. Seed the scaffolds with a neuronal cell suspension and incubate.

The coated surface and the scaffold pores provide a large surface area for high-density cell attachment and facilitate efficient nutrient and waste diffusion.

Remove the non-attached cells and add collagen, an extracellular matrix protein.

Incubate for collagen polymerization. This process embeds the cells within a gel-like 3D matrix on the porous scaffold.

Add media. Culture, and regularly replace half of the culture media to replenish nutrients without disturbing the cells.

Within the culture, the neuronal cell bodies remain anchored to the silk scaffold while the axons grow through the collagen matrix, forming 3D axonal networks.

This compartmentalized cell body and axonal localization results in the formation of a 3D brain-like polarized neural tissue model.

Inside a cell culture hood, place a pair of sterilized forceps, cell culture media, and the autoclaved scaffolds. Remove a 96-well plate from its wrapping and place inside. To