A neurorecording toolkit for longitudinal assessments of transplanted human cortical organoids <i>in vivo</i>

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 <i>in vivo</i>. 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 <i>in vivo</i> 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.