Defining minimum media requirements for ex vivo respiratory cilia studies using mouse tracheal tissue

Freshly harvested airway samples are routinely collected and imaged to study cilia function and diagnose motile ciliopathies. A previous study has suggested that Dulbecco's phosphate-buffered saline (DPBS) is just as effective as complex traditional cell culture media for the ex vivo assessment of cilia motility using mouse airway samples. The use of a less complex media to study cilia motility would be beneficial in reducing exogenous factors typically found in complex cell culture media that may influence cilia behaviour. Thus, the aim of this study was to determine the minimum media composition required to preserve airway ciliary function by incubating isolated mouse trachea sections in L15 and three simple saline-based solutions of increasing complexity (0.9% Saline, NaCl/KCl solution containing 150 mM NaCl/4.15 mM KCl, DPBS) following 0-12 hour storage at either 4 °C or 37.5 °C. Results reveal that a simple NaCl/KCl solution which approximates physiological extracellular concentrations of Na⁺, Cl^-, and K⁺ was sufficient to preserve ciliary motility in these tissues for up to 2 hours at 37.5 °C. Beyond 2 hours, samples stored in all solutions lacking an exogenous energy source displayed small but significant reductions in cilia beat frequency (CBF), which could be prevented by 10 mM glucose supplementation, maintaining CBF for up to 12 hours. In contrast, storage in 0.9% saline led to a rapid and significant impairment of cilia motility, with this impairment being significantly more pronounced following 4 °C storage vs. 37.5 °C storage. Additionally, ciliated cells were found to be more susceptible to storage associated injury and cell death than non-ciliated epithelial cells, with this vulnerability exacerbated by storage in 0.9% saline and at 4 °C. In conclusion, a simple NaCl/KCl solution is sufficient to maintain ciliary motility for up to 2 hours at 37.5 °C, with glucose supplementation alone required to preserve CBF for up to 12 hours. The use of simplified media for cilia motility studies provides a standardized and cost-effective foundation for future mechanistic studies allowing clearer insights into underlying pathways regulating motile cilia function.