A Technique to Generate Brain Organoids from Human Embryonic Stem Cells

Take colonies of human embryonic stem cells, or hESCs, cultured on a supporting matrix.

Aspirate the medium and add enzymes that degrade the matrix.

Wash to remove the enzymes. Then, add a medium and pipette to dislodge the loosened colonies, breaking them into smaller clusters.

Transfer the clusters into a fresh medium and incubate, allowing the clusters to form spherical aggregates.

Allow the spheres to settle and remove any dead cells.

Incubate with a medium containing basic fibroblast growth factor, or bFGF, for cell proliferation.

Replenish with media containing gradually decreasing bFGF concentrations to facilitate cell differentiation.

Introduce a neural induction medium, promoting hESC differentiation into neural stem cells or NSCs, transforming the spheres into organoids exhibiting a neural rosette-like structure.

Add antibiotics to prevent the growth of any contaminating microbes during prolonged culture.

The NSCs differentiate into neurons and glia, progressively developing the organoids into a complex structure resembling a brain cortex.