PDF, Englisch Download (36MB) | Nutzungsbedingungen

Abstract

In recent years, three-dimensional (3D) in vitro cell culture models such as spheroids and organoids have revolutionized life science research by providing a much more reliable context resembling the in vivo microenvironment. These systems yield important cell-to-cell interactions and induce cell differentiation. However, no conventional microscopy setup can provide sufficient imaging throughput as well as spatial and temporal resolution to enable full 3D live imaging and analysis down to subcellular processes. In this project, we established state-of-the-art light sheet microscopy for live, long-term imaging of a short interfering ribonucleic acid (siRNA) treated 3D cell culture model. Due to the high temporal and special resolution of the light sheet microscope, we minimized imaging artifacts and achieved unprecedented visual representations of spheroids throughout development and upon gene knock-down by siRNAs. Furthermore, we deployed a high-throughput image analysis pipeline and machine learning classification to evaluate global, cellular and subcellular features for a precise, quantitative gene knock-down phenotype description. The RNA interference (RNAi) induced gene knock-down phenotypes were replicated and compared by a novel molecular, site-specificepigenome modifying method. Throughout this project, we carefully evaluated every step of the workflow to improved its throughput and increased its reproducibility and usability. We addressed the key challenges in light sheet microscopy, such as sample preparation, data handling, image processing and analysis, thereby establishing quantitative light sheet microscopy screening of 3D cell culture models for many research applications. In total, we believe that our workflow can provide the basis for high-content analysis of 3D cell culture models for future research, enabling much more detailed functional experiments and basic research studies.