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

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.