In Situ 3D Super Resolution Nanoscale Imaging of Cell Spheroids by Expansion Microscopy

Three-dimensional (3D) cell models provide a physiologically relevant environment similar for improving drug screening. However, subcellular structure observation remains challenging due to light scattering. This study developed 3D HeLa cell spheroids in gelatin methacryloyl (GelMA) hydrogel as the in vitro 3D cell model for drug screening. A 4.6-fold expansion protocol enabled in situ high-resolution 3D mitochondrial imaging for cell spheroids cultured in GelMA. Notably, to our knowledge, this is the first instance of revealing mitochondrial morphology at the individual mitochondrial level within cell spheroids. Furthermore, quantitative analysis reveals the mitochondrial morphology change after drug treatment. This work establishes a high-content 3D cell model-based drug screening method, in which in situ subcellular structure imaging in 3D cell spheroids embedded in GelMA can be achieved with unprecedented resolution (about 90 nm) by commercial confocal microscope, facilitating a deeper understanding of drug mechanisms in cell spheroid-based drug screening.