목적: Olfactory organoids, 3D spheroid-structures derived from mouse adult tissue stem cells of the olfactory epithelium, have emerged as revolutionary models for understanding sensory neurobiology and host- pathogen interactions. In this study, we developed and characterized olfactory epithelial organoids with controlled polarity configurations, specifically apical-out and basal-out, to explore their functional implications in olfactory research. Moreover, we have established a method to reverse the polarity of the olfactory organoid so that the apical surface faces outward, maintaining the suspension structure while making the apical surface accessible to experimental challenges. 방법:Olfactory epithelial cells from 6-week-old mouse olfactory epithelium have been used to generate olfactory epithelial organoids. Cell proliferation was induced through Matrigel-embedded culture for 6 days and transferred to suspension culture for differentiation for 10 days. These organoids were characterized by immunostaining against olfactory epithelium cellular markers and by calcium imaging of responses to odors. Colony growth and sensory neuron maturation were investigated by quantitative PCR and immunostaining against neuronal sustentacular cell markers. 결과:Our meticulous investigation into the differentiation of type-specific cell localization in Matrigel-embedded and suspension-cultured organoids, through immunostaining against olfactory-related markers, has led to significant findings. The detection of the basal cell marker K5 in the inner region, the observation of Omp-positive cells in the outer region of apical-out organoids, and the confirmation that OSNs apically located of organoids respond to odorant stimulation through calcium imaging analysis, all stand as a testament to the thoroughness and validity of our research process. 결론:In summary, we identified heterogeneous cell types and differentiation of abundant mature olfactory sensory neurons(mOSNs) in olfactory epithelial organoids with apical-out polarity. These results suggest that the apical-out organoids developed a morphology similar to the mouse olfactory epithelial tissue structure. Furthermore, we propose that suspension-cultured organoids could be applied as a novel tool for olfactory research, including disease modeling, drug screening, host-pathogen interactions, and regeneration.