Intravital Multimodal Imaging of Human Cortical Organoid Transplantation in a Mouse Model of Chronic Stroke

ABSTRACT Chronic stroke poses enduring neurological deficits, while experimental strategies based on human induced pluripotent stem cell‐derived cortical organoids (COs) remain limited by the lack of tools for longitudinal in vivo graft monitoring. Here, we introduce the Multimodal Imaging Platform for Organoid Tracking (MIPOT), integrating MRI, bioluminescence imaging, surgical microscopy, and intravital two‐photon fluorescence microscopy to monitor CO transplantation in chronic stroke lesions in mice. MIPOT enabled confirmation of initial graft placement within cleaned infarct cavities, longitudinal assessment of viability‐associated bioluminescence signals, and high‐resolution visualization of graft‐derived cells and short‐term cellular dynamics. Bioluminescence imaging revealed a progressive decline in graft‐associated signals that stabilized at approximately 25% by two weeks, while endpoint histology confirmed limited persistence of human graft‐derived cells at later time points. Together, MIPOT provides a multimodal framework for monitoring organoid transplantation in the injured brain and supports future optimization of graft survival, maturation, and host–graft integration.