High-throughput 3D imaging flow cytometry of adherent 3D cell cultures

Three-dimensional (3D) cell cultures are indispensable in recapitulating in vivo environments. Among many 3D culture methods, the strategy to culture adherent cells on hydrogel beads to form spheroid-like structures is powerful for maintaining high cell viability and functions through an efficient supply of nutrients and oxygen. However, high-throughput, scalable technologies for 3D imaging of individual cells cultured on the hydrogel scaffolds are lacking. This study reports the development of a high-throughput, scalable 3D imaging flow cytometry (3D-iFCM) platform for analyzing spheroid models on hydrogel beads. This platform is realized by integrating a single objective lens-based fluorescence light-sheet microscopy with a microfluidic device employing a combination of hydrodynamic and acoustofluidic focusing techniques. This integration enabled an unprecedentedly high-throughput, robust optofluidic 3D imaging, processing 513 cells s-1 and a total of more than 104 cells within a minute. The large dataset obtained allows us to quantify and compare the nuclear morphology of adhering and suspended cells, revealing adhering cells have smaller nuclei with non-round surfaces. This platforms high throughput, robustness, and precision for analyzing the morphology of subcellular compartments in 3D culture models holds promising potential for various biomedical analyses, including image-based phenotypic screening of drugs with spheroids or organoids.