Fluorescence-based Single-cell Analysis of Whole-mount-stained and Cleared Microtissues and Organoids for High Throughput Screening

Three-dimensional (3D) cell culture, especially in the form of organ-like microtissues ("organoids"), has emerged as a novel tool potentially mimicking human tissue biology more closely than standard two-dimensional culture. Typically, tissue sectioning is the standard method for immunohistochemical analysis. However, it removes cells from their native niche and can result in the loss of 3D context during analyses. Automated workflows require parallel processing and analysis of hundreds to thousands of samples, and sectioning is mechanically complex, time-intensive, and thus less suited for automated workflows. Here, we present a simple protocol for combined whole-mount immunostaining, tissue-clearing, and optical analysis of large-scale (approx. 1 mm) 3D tissues with single-cell level resolution. While the protocol can be performed manually, it was specifically designed to be compatible with high-throughput applications and automated liquid handling systems. This approach is freely scalable and allows parallel automated processing of large sample numbers in standard labware. We have successfully applied the protocol to human mid- and forebrain organoids, but, in principle, the workflow is suitable for a variety of 3D tissue samples to facilitate the phenotypic discovery of cellular behaviors in 3D cell culture-based high-throughput screens. Graphic abstract: Automatable organoid clearing and high-content analysis workflow and timeline.