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To determine air-liquid interface (ALI) culture derived from cryopreserved mammalian tracheal ciliated cells is a viable ciliated cell model for the investigations of regulatory mechanisms of ciliary beat frequency (CBF), two studies were performed using ovine and porcine tracheae obtained from local slaughterhouses. The protease-digested tracheal ciliated cells were harvested and cultured at the ALI using collagen-coated, porous membrane inserts. In study 1, the ALI culturing protocols were established using non-cryopreserved ovine tracheal ciliated cells. Ciliogenesis was documented with immuno-histology and electron micrographs. Vigorous beating cilia were video-recorded. CBF was measured by laser light scattering. The functional integrity of the autonomic receptors of the ciliated cells was confirmed with the stimulatory responses of CBF using luminal methacholine and basolateral terbutaline. In study 2, porcine tracheal ciliated cells stored in liquid nitrogen for a minimum of four weeks were used. The cryopreserved cells were thawed and cultured using the ALI protocol established in study 1. After two months, cilia outgrowths were confirmed using video microscopy and scanning electron micrograph (SEM). The trans-epithelial resistances were 28.5 kΩ (n=4). Luminal applications of 1 µM and 10 µM methacholine stimulated CBF from a baseline of 7.4±0.2 Hz to 8.4±0.8 Hz and 7.7±0.4 Hz, respectively (n=5). Basolateral applications of 1 µM and 10 µM terbutaline stimulated CBF from a baseline of 7.5±0.3 Hz to 8.2±0.4Hz and 8.0±0.4 Hz, respectively (n=5). These data demonstrated that a ciliated cell bank can be established using cryo-preserved ciliated cells for pulmonary drug discovery and toxicological screening.

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