- Protocol for generating reproducible miniaturized controlled midbrain organoids.
Protocol for generating reproducible miniaturized controlled midbrain organoids.
STAR protocols (2023-07-23)
Muwan Chen, Jonathan Christos Niclis, Mark Denham
PMID37481727
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.
MATERIALS
Product Number
Brand
Product Description
Sigma-Aldrich
N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt, ≥96% (HPLC), powder
Sigma-Aldrich
Smoothened Agonist, SAG, A cell-permeable Smoothened Agonist, SAG, CAS 364590-63-6, modulates the coupling of Smo with its downstream effector by interacting with the Smo heptahelical domain (KD = 59 nM).
Sigma-Aldrich
Anti-Tyrosine Hydroxylase Antibody, clone LNC1, ascites fluid, clone LNC1, Chemicon®