NPC Neurosphere Assay: An In Vitro Assay to Assess the Migratory Response of Neural Precursor Cells from the Neurosphere

Neurospheres are non-adherent spherical aggregates of neural precursor cells or NPCs.

When plated under specific adherent culture conditions, NPCs may spontaneously migrate out from the neurosphere.

To assess the migratory response of NPCs, first, prepare an NPC suspension of desired density in a suitable media.

Next, seed the cell suspension into a media-containing culture plate and incubate.

The specific growth factors in the media support the proliferation of cells. Additionally, the absence of a coating substrate keeps the cells suspended in media, facilitating them to form 3D cell aggregates called neurospheres.

On attaining the desired size, collect the neurospheres. 

Centrifuge and resuspend the neurosphere pellet in the diluted media.

Then, plate the neurosphere suspension into an extracellular matrix-coated plate containing diluted media and incubate.

During culture, the neurospheres adhere to the extracellular matrix.

Over time, due to the constituents of the extracellular matrix, a few NPCs begin to migrate away from the neurosphere core.

Now, remove the media and fix the cells.

Capture phase-contrast images of the neurospheres exhibiting cell migration.

Using suitable software, measure the area of the total neurosphere and the inner cell mass.

Finally, subtract the inner cell mass area from the total neurosphere area to calculate the average NPC migration from the neurosphere.