Protocol for the establishment and morphological characterization of long-term cultivated murine cerebral organoids

Murine cerebral organoids provide a rapid and reproducible in vitro system that recapitulates key aspects of neurogenesis. While human cerebral organoid protocols are well established, methods for non-human models remain limited. Here, we present a protocol for generating long-term cultured murine cerebral organoids from E14.5 embryonic stem cells (ESCs). We describe steps for generating mature organoids, followed by histological processing including paraffin embedding and microtome sectioning. We then detail procedures for characterizing murine cerebral organoids through H&E staining and immunofluorescence techniques. • Optimized B27-guided protocol for long-term murine cerebral organoids • Stepwise maturation from neuroepithelial rosettes to cortical-like layers • Serial sectioning with histology and IF enables structural characterization • Murine cerebral organoids as a model to complement in vivo studies Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Murine cerebral organoids provide a rapid and reproducible in vitro system that recapitulates key aspects of neurogenesis. While human cerebral organoid protocols are well established, methods for non-human models remain limited. Here, we present a protocol for generating long-term cultured murine cerebral organoids from E14.5 embryonic stem cells (ESCs). We describe steps for generating mature organoids, followed by histological processing including paraffin embedding and microtome sectioning. We then detail procedures for characterizing murine cerebral organoids through H&E staining and immunofluorescence techniques.