Fluorescence-based Single-cell Analysis of Whole-mount-stained and Cleared Microtissues and Organoids for High Throughput Screening
Open accessAbstract
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.
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Specialized microscopy techniques and tissue processing (e.g., clearing) can overcome challenges in imaging 3D cultures, but they require additional specifications and 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).”
From Recommendations on fit-for-purpose criteria to establish quality management for microphysiological systems and for monitoring their reproducibility (2024) · cites this paper as Renner et al., 2021
The Abstract (page not provided) says whole-mount staining and tissue clearing enable imaging of entire 3D aggregates without sectioning. Day 6 (page not provided) specifies imaging settings that must be adjusted to the analysis, but the passages do not establish a requirement for additional quality control. (LLM, conf=0.84)
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Research works printed but not linked: 2
- Renner, H., Grabos, M., Becker, K. J., Kagermeier, T. E., Wu, J., Otto, M., Peischard, S., Zeuschner, D., TsyTsyura, Y., Disse, P., Klingauf, J., Leidel, S. A., Seebohm, G., Scholer, H. R. www.bio-protocol.org/e4050 Bio-protocol 11(12): e4050. DOI:10.21769/BioProtoc.4050
- Copyright Renner et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). Please cite this article as: Renner, H. et al., (2021). Fluorescence-based Single-cell Analysis of Whole-mount-stained and Cleared Microtissues and Organoids for High Throughput Screening. Bio-protocol 11(12): e4050. DOI: 10.21769/BioProtoc.4050.
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- Authors
- Renner, H., Otto, M., Grabos, M., Schöler, H., & Bruder, J.
- Affiliations
- Max Planck Institute for Molecular Biomedicine (DE)
- University of Münster (DE)
- Corresponding author
- Bruder, Jan
- Venue
- BIO-PROTOCOL
- Publisher
- Bio-Protocol, LLC
- Published
- 2021
- Volume
- 11
- Issue
- 12
- Pages
- e4050-e4050
- ISSN
- 2331-8325 (ISSN-L)
- PMC
- PMC8260262
- OpenAlex
- W3174159582
- Cited by
- 12
- References
- 13
- Citations by year
- 2026: 1 · 2025: 4 · 2024: 2 · 2023: 1 · 2022: 3 · 2021: 1
- FWCI
- 0.79
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- cc-by
- Locations
- BIO-PROTOCOL (publishedVersion, cc-by)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Funders
- International Max Planck Research School for Environmental, Cellular and Molecular Microbiology
- European Commission (669168, HORIZON2020)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
- Keywords
- organoids, microtissues, whole-mount immunostaining, tissue clearing, fluorescence imaging, single-cell analysis, high-throughput screening, automated liquid handling, 3D cell culture, brain organoids
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