The olfactory neuroepithelium (ON) collected with nasal swabs (NS) is a valuable source for studying brain processes in physiological and pathological conditions, due to its anatomical proximity to the brain and heterogeneous neural cell population. In this proof-of-concept study, we developed a protocol for co-culturing distinct cellular populations from ON, establishing a human olfactory neuroepithelium cell culture (hON) model from healthy individuals (HIs) without differentiation factors. We characterized the cellular composition of ON samples at harvesting, identifying sustentacular cells (Pan-CK⁺), neuronal cells (PGP9.5⁺), and ciliated respiratory cells (βIV-tubulin⁺) by immunofluorescence and flow cytometry. We assessed the expression and distribution of TDP-43, Tau, and α-synuclein, together with their phosphorylated isoforms, to establish a physiological reference profile. Furthermore, Seeding Amplification Assay (SAA) analysis, including disease-positive and healthy control cohorts, confirmed the absence of aggregated protein species in HIs. While p-Tau and p-α-syn localization was preserved between collection and culture, p-TDP-43 displayed neuron-specific nuclear redistribution upon culture, suggesting distinct regulatory dynamics. Longitudinal monitoring revealed that hON cultures maintain a stable neuronal population for up to 21 days, after which cells transition toward a fibroblast-like phenotype. This study establishes NS-derived hON as a feasible model for investigating neurodegenerative mechanisms, providing a baseline for disease comparisons.