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 seed the scaffolds, first, place one sterile scaffold per well in the 96-well plate. Add cell culture medium to immerse the scaffolds, and incubate at 37 degrees Celsius in a tissue culture incubator to equilibrate them for at least 30 minutes.

Following incubation, aspirate the excess medium from the scaffolds. Then, add 2 times 10 to the 6 rat cortical neuronal cells in 100 microliters of neurobasal medium per scaffold. Return the plate to the incubator, and leave overnight to allow the cells to attach to the scaffold. On the following morning, carefully aspirate the non-attached cells, and replace with 200 microliters of fresh cell culture medium. Incubate at 37 degrees Celsius for a brief period.

Next, aspirate the medium from the wells. Using sterile forceps, transfer the scaffolds containing cells to empty wells of the 96-well plate. Then to each scaffold, add 100 microliters of freshly diluted ice-cold 3 milligrams per milliliter rat tail collagen solution.

Return the cells to the 37 degrees Celsius incubator to allow polymerization of collagen for 30 minutes. Remove the plate from the incubator, and add 100 microliters of pre-warmed cell culture medium per well. Return the plate to the incubator for the desired period of cell growth, replacing half of the medium from each well with fresh medium every day for up to one week.