Understanding the complexities of the human brain and its associated disorders poses a significant challenge in neuroscience. Traditional research methods have limitations in replicating its intricacies, necessitating the development of in vitro models that can simulate its structure and function. Three-dimensional in vitro models, including organoids, cerebral organoids, bioprinted brain models, and functionalized brain organoids, offer promising platforms for studying human brain development, physiology, and disease. These models accurately replicate key aspects of human brain anatomy, gene expression, and cellular behavior, enabling drug discovery and toxicology studies while providing insights into human-specific phenomena not easily studied in animal models. The use of human-induced pluripotent stem cells has revolutionized the generation of 3D brain structures, with various techniques developed to generate specific brain regions. These advancements facilitate the study of brain structure development and function, overcoming previous limitations due to the scarcity of human brain samples. This technical review provides an overview of current 3D in vitro models of the human cortex, their development, characterization, and limitations, and explores the state of the art and future directions in the field, with a specific focus on their applications in studying neurodevelopmental and neurodegenerative disorders.

Human induced pluripotent stem cell (iPSC)-derived neural organoids have emerged as valuable models for investigating human neurodevelopment and neurological disorders. However, their complex three-dimensional architecture poses significant challenges for conventional histological approaches, which rely on physical sectioning and inevitably disrupt spatial relationships within the tissue. Volumetric imaging of intact organoids typically requires light-sheet fluorescence microscopy, a technology not widely accessible to standard cell biology laboratories. Here, we show that solvent-based tissue clearing, using the iDISCO+ and Visikol^® HISTO protocols, combined with conventional laser-scanning and spinning-disk confocal microscopy, platforms already available in most imaging facilities, is sufficient to resolve neuroepithelial rosette-like structures, neuronal networks, and astroglial components within intact human cortical and retinal organoids, while preserving immunofluorescent labeling and tissue architecture. The workflow was also compatible with commonly used immunofluorescence markers. Although light-sheet fluorescence microscopy remains advantageous for large-scale whole-sample imaging, our results show that cleared human organoids within the size range analyzed here can be effectively visualized using accessible confocal systems. This study provides a practical strategy for three-dimensional imaging of intact human neural organoids, facilitating spatial analysis of developmental organization and disease-relevant phenotypes in laboratories without dedicated light-sheet microscopy infrastructure.