Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward

Frontiers in Molecular Biosciences
Elina NürnbergRüdiger Rudolf

Abstract

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. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. 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. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. 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. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing prot...Continue Reading

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Citations

Mar 29, 2020·Sensors·Elena von MolitorTiziana Cesetti
Dec 10, 2020·Pharmaceutics·Bárbara PintoHassan Bousbaa
Feb 11, 2021·In Vitro Cellular & Developmental Biology. Animal·Terry Riss, O Joseph Trask
Feb 17, 2021·Methods and Applications in Fluorescence·Sergiy V Avilov
Mar 16, 2021·Computational and Structural Biotechnology Journal·Akos DiosdiPeter Horvath
Jul 2, 2021·Frontiers in Cell and Developmental Biology·Etsuo A Susaki, Minoru Takasato
Sep 3, 2021·Chaos·Nadezhda Semenova, Valery V Tuchin

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Methods Mentioned

BETA
light sheet microscopy
light scattering
fluorescence imaging
chips
confocal microscopy
chip

Software Mentioned

eS
uDISCO
iDISCO
FIJI
Leica Application Suite X
Clear
Sca
RapiClear
3DISCO
CytoVista

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