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

Abstract

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.

Recommendations on fit-for-purpose criteria to establish quality management for microphysiological systems and for monitoring their reproducibility

“…It could be difficult to use these readouts for QC. Specialized microscopy techniques and tissue processing (e.g., clearing) can overcome these challenges, but they require additional specifications and QC ( Nurnberg et al., 2020 ; Renner et al., 2021 ; Cheng et al., 2023 ; Yoshida et al., 2020 ; Kahn-Krell et al., 2022 ).…”

Three-Dimensional Cell Culture Systems in Pediatric and Adult Brain Tumor Precision Medicine

“…Automating organoid culture is a promising approach to increase reproducibility through a more homogeneous and standardized workflow, which also makes the systems scalable for high-throughput screening strategies. Recently, Renner et al succeeded in fully automating mid- and forebrain organoid generation, culture, and optical analysis [ 109 , 110 ] in a 96-well format. Their automated midbrain organoids were highly homogeneous and reproducible in size, marker gene expression, and transcriptional levels [ 109 ].…”

“…Their automated midbrain organoids were highly homogeneous and reproducible in size, marker gene expression, and transcriptional levels [ 109 ]. Furthermore, they established a high-throughput screen that assessed different neuronal subpopulations while retaining their spatial information through automated whole-mount immunostaining, tissue clearing, and image analysis [ 109 , 110 ]. Translating other current 3D brain tumor models to an automated workflow could further increase the homogeneity and reproducibility of cancer organoid approaches.…”