A fully automated high throughput-workflow for human neural organoids

Recent advances in stem cell technology have led to the development of three-dimensional (3D) culture systems called organoids, which have fueled hopes to bring about the next generation of more physiologically relevant high throughput screens (HTS). However, the adaptation of established organoid protocols for HTS applications has so far been elusive. Here, we present a fully scalable, HTS-compatible workflow for the automated generation, maintenance, whole mount staining, clearing, and optical analysis of human neural organoids generated from neural precursor cells in a standard 96-well format. By combining organoid generation and analysis steps in an automated fashion, we can perform quantitative whole-organoid high content imaging with single cell resolution. The resulting organoids are highly homogeneous with regard to their morphology, size, global gene expression, cellular composition, and structure. Calcium imaging suggests organoid-wide synchronized functional coupling. The scalability of our approach has the potential to form the basis for 3D tissue-based screening in a variety of applications including drug development, toxicology studies, and disease modeling. Overall design: Comparison of automated midbrain organoids and established induced plurioptent stem cell (iPSC)-derived organoids through RNA sequencing.