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bioRxiv · 10.1101/2023.07.10.548361

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

Yamashita, M.·Tamamitsu, M.·Kirisako, H.·Goda, Y.·Chen, X.·Hattori, K.·Ota, S.
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Abstract

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.

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Yamashita, M., Tamamitsu, M., Kirisako, H., Goda, Y., Chen, X., Hattori, K., Ota, S.. 2023-07-11. High-throughput 3D imaging flow cytometry of adherent 3D cell cultures. https://doi.org/10.1101/2023.07.10.548361

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