Abstract
Here, we present a protocol for generating miniaturized controlled midbrain organoids (MiCOs) of reproducible size and cellular composition, without a necrotic center. We describe steps for maintaining and passaging human pluripotent stem cells, generating MiCOs using AggreWell™400, and maintaining them in an EB-Disk360on an orbital shaker, eliminating the need for Matrigel or a spinner flask and preventing organoid fusion. We then detail organoid collection for different endpoint analysis. This protocol is suitable for compound screening and disease modeling studies.