3D quantitative assessment for nuclear morphology in osteocytic spheroid with optical clearing technique

In recent years, three-dimensional (3D) cell culture has been attracting attention as a cell culture model that mimics an environment closer to that of a living organism. It is known that there is a close relationship between cell nuclear shape and cellular function, which highlights the importance of cell nucleus shape analysis in the 3D culture. On the other hand, it is difficult to observe the cell nuclei inside the 3D culture models because the penetration depth of the laser light under a microscope is limited. In this study, we adopted an aqueous iodixanol solution to the 3D osteocytic spheroids derived from mouse osteoblast precursor cells to make the spheroids transparent for 3D quantitative analysis. With a custom-made image analysis pipeline in Python, we found that the aspect ratio of the cell nuclei near the surface of the spheroid was significantly greater than that at the center, suggesting that the nuclei on the surface were deformed more than those at the center. The results also quantitatively showed that the orientation of nuclei in the center of the spheroid was randomly distributed, whereas those on the surface of the spheroid were oriented parallel to the surface of the spheroid. Our 3D quantitative method with an optical clearing technique will contribute to the 3D culture models including various organoid models to elucidate the nuclear deformation during the development of the organs. Insight box Although 3D cell culture has been a powerful tool in the fields of fundamental biology and tissue engineering, it raises the demand for quantification techniques for cell nuclear morphology in the 3D culture model. In this study, we attempted to optically clear a 3D osteocytic spheroid model using iodixanol solution for the nuclear observation inside the spheroid. Moreover, using a custom-made image analysis pipeline in Python, we successfully quantified the nuclear morphology regarding aspect ratio and orientation. Our quantitative method with the optical clearing technique will contribute to the 3D culture models such as various organoid models to elucidate the nuclear deformation during the development of the organs.