Generation and Maintenance of Homogeneous Human Midbrain Organoids.

Three-dimensional cell cultures ("organoids") promise to better recapitulate native tissue physiology than traditional 2D cultures and are becoming increasingly interesting for disease modeling and compound screening efforts. While a number of protocols for the generation of neural organoids have been published, most protocols require extensive manual handling and result in heterogeneous aggregates with high sample-to-sample variation, which can hinder screening-based strategies. We have now developed a fast and efficient protocol for the generation and maintenance of highly homogeneous and reproducible midbrain organoids. The protocol is streamlined for use in fully automated workflows but can also be performed manually without the need for highly specialized equipment. It relies on the aggregation of small molecule neural precursor cells (smNPCs) in standard 96-well V-bottomed plates under static culture conditions without cumbersome matrix embedding. The result is ready-to-assay uniform 3D human midbrain organoids available in freely scalable quantities for downstream analyses in 3D cell culture. Graphic abstract: Automated midbrain organoid generation workflow and timeline.