Amongst others, hydrogels, decellularized matrices, porous polymers, and nanofibers might serve as scaffolds in static or dynamic experimental setups can be designed ( Das et al., 2015 Carvalho et al., 2017), e.g., in organ-on-a-chip systems ( Bauer et al., 2018 Hübner et al., 2018). During the last decade, there has been a substantial increase in the use of three-dimensional (3D) cell culture models in a large variety of biological fields, ranging from developmental biology ( Lancaster et al., 2013) to oncology ( Fong et al., 2016 Drost and Clevers, 2018) and drug discovery ( Alepee et al., 2014).Ĭoarsely, 3D- in vitro models can be divided into matrix-supported and matrix-free models ( Wang et al., 2014). The absence of nutrient and oxygen gradients, as well as restricted migration potential grown on a plastic surface, further contribute to a limited representation of physiology in 2D in vitro systems ( Duval et al., 2017).
In two-dimensional (2D) cell culture models, the lack of comprehensive interaction among cells via cell–cell-contacts and between cells with their surrounding extracellular matrix can lead to non-physiological morphology, gene expression, and cellular behavior ( Zschenker et al., 2012 Luca et al., 2013). Since in vitro monolayer cell cultures do not sufficiently reflect this attribute, they have often been considered to be limited in representing the physiology of organs and tissues ( Imamura et al., 2015 Hafner et al., 2017).
In the human body, cells grow in clusters, organizing themselves into function-specific tissues and multifunctional organs in all three spatial dimensions. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols – in particular, for screening purposes – clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Third, z-compensation was imperative to minimize variations in signal-to-noise ratio. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid- and chip-based three-dimensional cell cultures of approximately 300 μm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. 4TIP Oncology, Merck Healthcare KGaA, Darmstadt, Germany.2Zentralinstitut für Seelische Gesundheit, Department of Translational Brain Research, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.1Institute of Molecular and Cell Biology, Faculty of Biotechnology, Mannheim University of Applied Sciences, Mannheim, Germany.Elina Nürnberg 1,2†, Mario Vitacolonna 1†, Julia Klicks 1, Elena von Molitor 1, Tiziana Cesetti 1, Florian Keller 1, Roman Bruch 1, Torsten Ertongur-Fauth 3, Katja Riedel 3, Paul Scholz 3, Thorsten Lau 2, Richard Schneider 4, Julia Meier 4, Mathias Hafner 1 and Rüdiger Rudolf 1*
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