Multiscale Analysis of Cellular Composition and Morphology in Intact Cerebral Organoids

Abstract

Cerebral organoids recapitulate in vivo phenotypes and physiological functions of the brain and have great potential in studying brain development, modeling diseases, and conducting neural network research. It is essential to obtain whole-mount three-dimensional (3D) images of cerebral organoids at cellular levels to explore their characteristics and applications. Existing histological strategies sacrifice inherent spatial characteristics of organoids, and the strategy for volume imaging and 3D analysis of entire organoids is urgently needed. Here, we proposed a high-resolution imaging pipeline based on fluorescent labeling by viral transduction and 3D immunostaining with fluorescence micro-optical sectioning tomography (fMOST). We were able to image intact organoids using our pipeline, revealing cytoarchitecture information of organoids and the spatial localization of neurons and glial fibrillary acidic protein positive cells (GFAP+ cells). We performed single-cell reconstruction to analyze the morphology of neurons and GFAP+ cells. Localization and quantitative analysis of cortical layer markers revealed heterogeneity of organoids. This pipeline enabled acquisition of high-resolution spatial information of millimeter-scale organoids for analyzing their cell composition and morphology.

Promising Prospects for Human Cerebral Organoids to Advance Alzheimer's Disease Research

“…As explained above, in vitro 3D models have compensated for the shortcomings of previous models, providing a more reliable view of cell interaction, drug response, and spatiotemporal development. Other advantages are that it can allow us to obtain organoids from patients for more personalized treatment [82] whole organoid image analyses can be performed for a global understanding [143]. It also allows us to reproduce and study the extracellular environment [100], how to use them for the creation of virtual platforms, allowing us to test new strategies without long protocols or ethical conflicts [118,144,145], or delving into stem cell therapy [146].…”

Cerebral Organoid Arrays for Batch Phenotypic Analysis in Sections and Three Dimensions

“…As organoid culturing techniques developed, some cerebral organoids with high complexity reached several millimeters in diameter, posing a challenge for in situ imaging and phenotypic analysis of organoids [ 12 , 13 ]. As a result, we previously developed a whole-organoid processing pipeline for labeling, embedding, imaging, and analyzing intact millimeter-scale cerebral organoids, which utilized the fMOST system to produce high-resolution 3D datasets, where we also demonstrated the neural rosette structures present in commercial cerebral organoids [ 14 ]. In addition to the large size of cerebral organoids, batch processing of organoids is another challenge in the field due to the inherent variability among individuals and batches of cerebral organoids [ 15 ].…”