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

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.