This article describes a method for generating cerebellar organoids from human induced pluripotent stem cells (iPSCs) using a bioreactor system. The process involves culturing iPSCs, promoting neural differentiation, and maturing cerebellar progenitors into functional neurons.
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.
On day 1, with day 0 being the day of seeding the bioreactor, place the bioreactor and the base unit in a sterile flow hood. Then, use a serological pipette to collect a 1-milliliter sample of the cell suspension. Plate the cell suspension in an ultra-low attachment 24-well plate. Use a microscope to confirm that iPSC-derived aggregates have formed. If so, capture images of the aggregates at 40x or 100x.
Continue culturing the iPSCs in the bioreactor until the average diameter of the aggregates is 100 micrometers. Then, replace 80% of the medium with fresh mTesR1 without ROCK inhibitor. When the aggregates reach 200 to 250 micrometers in diameter, let all the organoids settle at the bottom of the bioreactor. Then, replace all the spent medium with gfCDM differentiation medium.
Place the base unit and the bioreactor in the incubator and decrease agitation to 25 RPM.
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