Establishment of in vitro nasal epithelial model for alternative toxicity test

Although there are strong urge to restrict the animal experiments for toxicity test, the alternative in-vitro test model or guideline is not yet been established. The present study attempted to establish the in-vitro nasal epithelial model for alternative toxicity test. Human nasal epithelial(HNE) cells were isolated by dispase II treatment, and cultured monolayer until confluent. HNE cells were harvested and seeded onto 12 mm millicell. The cells were cultured for 7 days of submerged condition and following 7 days of air-liquid interface(ALI) condition. Fully differentiated cell sheets were prepared 0.4 μm sections, and H&E and immunohistochemical staining were compared with intact human nasal epithelium and conjunctiva. Primary cultured HNE cells showed cobblestone-like morphology without contamination with other cell types. Histological observation showed well differentiated ciliogenesis. The expressions of p63 at basal cell nucleus, CD44v6 at cell-cell junctions of basal cell layer, Na⁺/K⁺ ATPase at apical layer was observed. Also ColIV, LN5, CK3/12, CK13, CK5, MUC1, and MUC5AC were positively expressed. These results suggest that in-vitro differentiated nasal epithelial model is suitable for alternative toxicity test due to its similarity with nasal epithelium. Taken together, our nasal epithelial model will be useful for alternative toxicity test.