Klorfeld-Auslender, S. | Search bioRxiv

bioRxiv2026

Seeing clearly with CLARI-O: a window into cellular architecture, interactions, and morphology of organoid models.

Cortical organoids (COs) represent a powerful in vitro model system that recapitulates key aspects of human brain development, enabling the study of neurodevelopmental processes, cellular diversity, and disease mechanisms in a physiologically relevant 3D environment. However, traditional histological analysis of COs relies on tissue sectioning, which limits the ability to capture the full spatial complexity of organoid architecture. In this study, we establish a framework for applying CLARI-O, an improved tissue-clearing technique, for intact COs and organoid-based systems, enabling comprehensive 3D visualization and analysis of 3D organizational features. Using CLARI-O in combination with high-resolution imaging, we demonstrate the utility of tissue clearing for studying glial populations, including oligodendrocytes and microglia, considered to be underrepresented in COs, and their interactions with neurons. Additionally, we apply this method to forebrain assembloids (FAs) to visualize cellular heterogeneity and the interface between ventral and dorsal regions. Finally, we use CLARI-O to study mouse brains containing xenotransplanted COs (MB-COs) to evaluate human cell integration, migration, vascularization, and structural connectivity. This is the first study to demonstrate how tissue clearing can be used after functional assays such as calcium imaging to correlate neural activity with post hoc structural analysis in MB-COs. Together, this work establishes CLARI-O as a powerful tool for advancing 3D structural and functional interrogation of human CO-derived systems, enhancing their value for disease modeling, drug screening, and translational neuroscience. MotivationCortical organoids have become an increasingly powerful tool in neuroscience. Their complexity has expanded substantially, now incorporating exogenous lineages, fusing organoids with distinct regional identities (assembloids), and enabling xenotransplantation into in-vivo environments. These advancements require more sophisticated technological approaches that are capable of capturing the intricate three-dimensional cyotarchitecture and organization of intact organoid systems both in vitro and after xenotransplantation in vivo. Tissue-clearing methodologies offer a unique opportunity to visualize these structural and cellular features with exceptional depth and resolution. Graphical abstract HighlightsO_LIWe optimized clearing protocols to develop an organoid specific clearing method (CLARI-O) that enables high-resolution visualization of diverse neuronal and glial populations without tissue sectioning, preserving long-range connections and cellular processes. C_LIO_LIForebrain assembloids used to study neuronal and oligodendrocyte migration can be effectively processed using CLARI-O, allowing detailed visualization of fusion interface. C_LIO_LIWe established a robust framework for CLARI-O-based clearing of mouse brain tissue containing xenotransplanted human cortical organoids, enabling comprehensive 3D analysis of graft development, integration, and vascularization in vivo. C_LI

neuroscience↗

bioRxiv2025

A neurorecording toolkit for longitudinal assessments of transplanted human cortical organoids in vivo

Human cortical organoids (hCOs) are three-dimensional neural cell aggregates that recapitulate certain structural and functional aspects of the developing human cortex. Xenotransplantation of hCOs into the rodent brain enables human-centric modeling of neurodevelopmental processes in a physiologically relevant environment. Here, we present a neurorecording toolkit for longitudinal structural and functional assessment of hCO xenografts as they mature in vivo. Single hCOs were implanted into the retrosplenial cortex of adult immunodeficient mice and monitored for up to 8 months. Optical coherence tomography was used for label-free imaging of xenograft vascularization and structure, enabling quantitative assessments of capillary density and graft volume. To probe neuronal activity, human neurons were labeled with a calcium sensor before implantation using either adeno-associated or lentivirus for sparse or dense neuronal labeling, respectively. Fluorescent imaging was conducted using two-photon, widefield, and swept confocally-aligned planar excitation microscopy for single cell, whole-graft, and volumetric calcium imaging, respectively. Results from these modalities indicate an increase in neuronal activity and synchronicity over time during in vivo graft maturation. Further, we chronically implanted surface graphene microelectrode arrays (gMEAs) and performed recordings of xenograft and host local field potential signals simultaneously with 2P calcium imaging, confirming the spatial localization and human origin of electrical signals recorded at the xenograft surface.

neuroscience↗