Generation of Spheroids from Human Pluripotent Stem Cells
Transcrição
Begin with colonies of human pluripotent stem cells or hPSCs attached to a suitable substrate in a culture medium.
Remove the medium. Add a neuronal medium containing specific inhibitors and incubate.
These inhibitors direct the maturation of hPSCs toward a neuronal lineage, forming neuroectodermal colonies. These are precursors to small, spherical structures called spheroids.
Remove the medium and wash with a salt buffer.
Incubate with a digestive enzyme to detach the colonies from the plate.
Add the neuronal medium to stop the enzymatic activity.
Collect the colonies and transfer them to a tube.
Allow them to settle, and then discard the supernatant.
Resuspend the colonies in a neuronal medium containing a growth factor.
Transfer the colonies to a multi-well plate and incubate.
This growth factor facilitates the self-organization and multiplication of neuroectodermal cells, leading them to form a spheroid.
