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

Area of Science:

• Biomedical Imaging
• Cell Biology
• Biotechnology

Background:

• Three-dimensional (3D) cell cultures like spheroids and organoids are vital research models.
• Optical tissue clearing improves visualization of these complex biological structures.
• These models are increasingly used for drug screening and fundamental research.

Purpose of the Study:

• To systematically evaluate six optical tissue clearing/embedding protocols.
• To assess protocol applicability on seven types of 3D cell cultures (spheroids and chip-based).
• To quantify the impact of clearing on fluorescence imaging and subsequent analysis.

Main Methods:

• Comparison of six clearing/embedding protocols on 3D cell cultures (~300 μm).
• Staining with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein.
• Whole mount confocal microscopy and semi-automated image analysis for quantification.

Main Results:

• Significant variability in transparency, shrinkage, and dye quenching across protocols.
• Clearing protocols were more efficient for single-cell-type samples than co-cultures.
• Z-compensation is crucial for maintaining signal-to-noise ratio.
• Cell density, shrinkage, and signal uniformity impact segmentation and cell counting.
• 88% Glycerol demonstrated the most promising balance of effectiveness, simplicity, and speed.

Conclusions:

• Optical tissue clearing methods vary significantly in their effects on 3D cell cultures.
• Protocol choice impacts imaging quality, data segmentation, and quantitative analysis.
• 88% Glycerol emerges as a highly promising clearing agent for 3D cell culture imaging, particularly for high-throughput screening.