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

JoVE Encyclopedia of Experiments

Neurowetenschap

0 weergaven • 2:51 min • July 8th, 2025

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Begin with a multi-well plate containing a porous insert that holds a polystyrene scaffold possessing a three-dimensional porous structure.

Add ethanol to rehydrate the scaffold and wash it with the medium.

Introduce a medium containing an extracellular matrix protein, laminin. Incubate to allow laminin to coat the scaffold.

Seed the human neural stem cells in a neural medium containing growth factors onto the laminin-coated scaffold.

Incubate to allow the stem cell migration into the porous scaffold where the laminin promotes cell adherence to the scaffold.

The cells utilize the growth factors and proliferate within this scaffold.

Add a growth factor-free neural medium and regularly replace the spent medium to maintain cell viability.

The absence of growth factors stimulates the differentiation of stem cells into neural progenitor cells.

Over time, the neural processes of these progenitor cells elongate, allowing multidirectional cell growth and forming a three-dimensional culture.

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.

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Laatst bijgewerkt: 1 augustus 2026