Generating Mature Cerebellar Organoids From Induced Pluripotent Stem Cells Using Single-Use Bioreactors

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.