Differentiating Human Neuronal Stem Cells into 3D Culture Using a Porous Scaffold

Rehydrate the scaffolds by adding to each well of the Alvetex 12-well plate, 2 milliliters of 70% ethanol, prepared using water for irrigation. Avoid touching the scaffolds by dispensing the 70% ethanol down the wall of each well. Carefully aspirate the 70% ethanol and immediately wash the scaffolds with 2 milliliters per well of DMEM F12 for one minute.

Repeat the wash procedure twice. Carefully aspirate the DMEM F12 after the final wash. Coat the scaffolds by adding 2 milliliters of laminin in DMEM F12 to each well. Incubate the plate at 37 degrees Celsius in a 5% carbon dioxide humidified incubator for a minimum of two hours.

Next, wash the plate with warm DMEM F12. Seed each scaffold with approximately 500,000 human neural stem cells or HNSCs in a volume of 150 microliters of RMM medium with growth factors. Incubate the plate for three hours to allow the cells to settle into the scaffolds.

After three hours, add 3.5 milliliters of RMM to each well, taking care not to dislodge cells from the scaffolds. Incubate the plate for seven or 21 days. Replace the medium after one day, which is the first feeding, and replace the medium again 2 to 3 days after the first feeding.