Protocol for culturing olfactory epithelium organoids supporting neuronal differentiation

The olfactory epithelium (OE) contains stem cells capable of generating olfactory sensory neurons throughout life, making it a valuable model for studying epithelial neurogenesis. We present a protocol to develop a highly robust 3D mouse organoid model from OE cells. We describe a workflow for dissecting murine OE and subsequent organoid culturing. We also provide guidance on how to perform immunostaining of the organoids. This protocol has been validated for mice and does not require fluorescence-activated cell purification. For complete details on the use and execution of this protocol, please refer to Gameiro et al. 1 • Instructions for mouse olfactory epithelium harvesting • Steps for developing a 3D but adherent mouse olfactory epithelium organoids • Guidance for immunostaining of olfactory epithelium organoids Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The olfactory epithelium (OE) contains stem cells capable of generating olfactory sensory neurons throughout life, making it a valuable model for studying epithelial neurogenesis. We present a protocol to develop a highly robust 3D mouse organoid model from OE cells. We describe a workflow for dissecting murine OE and subsequent organoid culturing. We also provide guidance on how to perform immunostaining of the organoids. This protocol has been validated for mice and does not require fluorescence-activated cell purification.