Generation of Spheroids from Human Pluripotent Stem Cells

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.

To begin, plate the hPSC colonies on an hESC-qualified basement membrane matrix from one well of a 6-well plate at 60% confluency into three wells to achieve 20% to 30% density, and then, proceed with induction of 2D neuroectodermal colonies.

Add fresh N2 media supplemented wit