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Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids
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Integrated light and electron microscopy workflow for morphological, molecular and ultrastructural analysis of
Larisa Tratnjek1, Aleksandar Janev1, Nataša Resnik1
1Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Plos One
|March 3, 2026
Summary
Researchers developed microscopy protocols for characterizing 3D cancer spheroids. These methods enable detailed analysis of spheroid morphology and protein distribution, advancing cancer research and drug development.
Area of Science:
- Biomedical Engineering
- Cancer Research
- Microscopy
Background:
- Traditional 2D cell cultures inadequately model tumor complexity.
- Three-dimensional (3D) spheroids offer a more accurate in vitro cancer model, mimicking in vivo tumor microenvironments.
- Comprehensive characterization of spheroids is crucial for understanding cancer progression and treatment response.
Purpose of the Study:
- To establish detailed light and electron microscopy protocols for comprehensive spheroid analysis.
- To enable accurate morphological, molecular, and ultrastructural characterization of 3D cancer models.
- To provide a versatile toolkit for spheroid analysis adaptable to various cell types.
Main Methods:
- Development of protocols for light microscopy (histological staining, immunofluorescence) via cryosectioning and paraffin embedding.
- Application of whole-mount immunofluorescence with optical clearing and confocal microscopy for protein localization.
- Utilization of scanning and transmission electron microscopy for high-resolution surface and ultrastructural analysis.
Main Results:
- Optimized protocols allow detailed visualization of spheroid morphology, protein expression, and cellular organization.
- Integrated microscopy workflows provide complementary insights into spheroid characteristics.
- Demonstrated adaptability of protocols for urothelial cell line spheroids (SV-HUC-1 and T24).
Conclusions:
- The developed integrated microscopy workflow enables thorough and accurate characterization of 3D cancer spheroids.
- These protocols establish a comprehensive baseline for future functional studies of cancer models.
- The adaptable methods support a broad range of applications in cancer research and drug discovery.
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