Generating Mature Cerebellar Organoids From Induced Pluripotent Stem Cells Using Single-Use Bioreactors
Transcrição
Seed human induced pluripotent stem cells, or iPSCs, in a bioreactor. The iPSCs are resuspended in media containing a cellular apoptosis inhibitor.
Under a magnetic field, the vertical wheel of the bioreactor rotates at a controlled speed, facilitating cell-cell contact and aggregate formation. Remove most of the media and add fresh media without the inhibitor.
Continue culturing. Once the aggregates reach an optimal size, allow them to settle by gravity. Remove the medium.
Add differentiation media containing growth factors, and culture with a reduced bioreactor wheel speed. The growth factors promote neural differentiation within the aggregates.
Next, add fresh medium supplemented with growth factors and chemokines that induce the development of cerebellar progenitors.
Gradually, the cerebellar progenitors organize into distinct layers within the aggregates, forming early cerebellar organoids.
Finally, add a neuronal medium containing specific neurotrophic factors.
These factors induce cerebellar progenitor maturation into synaptically active functional cerebellar neurons, generating mature organoids.
