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Abstract
Three-dimensional (3D) culture systems have fueled hopes to bring about the next generation of more physiologically relevant high-throughput screens (HTS). However, current protocols yield either complex but highly heterogeneous aggregates ('organoids') or 3D structures with less physiological relevance ('spheroids'). Here, we present a scalable, HTS-compatible workflow for the automated generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates. The resulting organoids possess a highly homogeneous morphology, size, global gene expression, cellular composition, and structure. They present significant features of the human midbrain and display spontaneous aggregate-wide synchronized neural activity. By automating the entire workflow from generation to analysis, we enhance the intra- and inter-batch reproducibility as demonstrated via RNA sequencing and quantitative whole mount high-content imaging. This allows assessing drug effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow.
Highlights
A number of uniquely human diseases, including Parkinson’s disease, would greatly benefit from a comprehensive human cellular in vitro model that recapitulates key characteristics of midbrain tissues in a high-throughput-compatible format
In order to limit cellular heterogeneity during differentiation, we produced human automated midbrain organoids (AMOs) starting from small molecule neural precursor cells (Reinhardt et al, 2013a), which in turn originate from pluripotent stem cells (PSCs)
Since homogeneity and reproducibility are crucial for screening applications, we examined the variance of AMOs on the gene expression level and compared it to that of published midbrain organoids (Jo et al, 2016)
Summary
A number of uniquely human diseases, including Parkinson’s disease, would greatly benefit from a comprehensive human cellular in vitro model that recapitulates key characteristics of midbrain tissues in a high-throughput-compatible format. The robots perform a series of precisely controlled tasks – including dispensing the initial cells into wells, feeding organoids as they grow and testing them at different stages of development These mini-brains, which are the size of the head of a pin, mimic the part of the brain where Parkinson’s disease first manifests. The resulting AMOs are similar to published midbrain organoids with regard to their expression of midbrain-specific markers and cell populations, yet maintain a reproducible homogeneous phenotype They mimic relevant organ function in the form of spontaneous, highly synchronized neural activity indicating functional cellular coupling across the entire AMO. Their high homogeneity, reproducibility, culture format, and fast development of approximately one month render them ideal for high-throughput screening applications. Our automation of the entire workflow from seeding to analysis in standard plates allows for easy scale-up and implementation into existing screening facilities
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