Culture and differentiation of mouse tracheal epithelial cells at air-liquid interface / 中国药理学通报

ABSTRACT

Aim Air-liquid interface (ALI) cultures of mouse tracheal epithelial cells (MTEC) are a well-established model to study airway epithelial cells. MTEC provides a powerful ap¬proach for the evaluation of the inhalation toxicological in vitro. Methods C57BL/6 mouse tracheal-bronchial epithelial cells were obtained by digestion with protease in cold temperature o- vemight, and the digestion time was optimized to ensure the quantity and viability of the obtained cells. The cells were cul¬tured into collagen coated Transwell inserts. Proliferating phase and air-liquid interface culture were promoted with different cul¬ture media. The expression of tight junction protein and cell trans-epithelial electrical resistance(TEER) were used to evalu¬ate the formation of tight junction between cells and the analysis of cell polarity. The cilia structure was confirmed by electron mi¬ croscopy and immunofluorescence. Results Highly purified and viable primary airway epithelial cells could be harvested and subcultured by our methods, including morphology and immuno- cytochemistry staining confirmed the expression of MUC5AC, a- tubulin, p-tubulin-IV and ZO-1. The development of tight-junc¬tions and epithelium were similar with pseudostratified ciliated columnar epithelium morphology. Conclusions A comprehen¬sive protocol for ALI culture was established, reproducing the characteristic pseudostratified ciliated columnar epithelium mor¬phology and physiological functions in vitro. The MTEC protocol provides a stable and reliable method for the isolation, mucocili¬ary differentiation and reproducing.