2Columbia University, New York, NY
3University of Pittsburgh, Pittsburgh, PA, USA
Air-liquid interface models using murine tracheal respiratory epithelium have revolutionized the in vitro study of pulmonary diseases. This model is often impractical because of the small number of respiratory epithelial cells that can be isolated from the mouse trachea. We describe a simple technique to harvest the murine nasal septum and grow the epithelial cells in an air-liquid interface. The degree of ciliation of mouse trachea, nasal septum, and their respective cultured epithelium at an air-liquid interface were compared by scanning electron microscopy (SEM). Immunocytochemistry for type IV β-tubulin and zona occludens-1 (Zo-1) are performed to determine differentiation and confluence, respectively. To rule out contamination with olfactory epithelium (OE), immunocytochemistry for olfactory marker protein (OMP) was performed. Transepithelial resistance and potential measurements were determined using a modified vertical Ussing chamber. SEM reveals approximately 90% ciliated respiratory epithelium in the nasal septum as compared with 35% in the mouse trachea. The septal air-liquid interface culture demonstrates comparable ciliated respiratory epithelium to the nasal septum. Immunocytochemistry demonstrates an intact monolayer and diffuse differentiated ciliated epithelium. These cultures exhibit a transepithelial resistance and potential confirming a confluent monolayer with electrically active airway epithelium containing both a sodium-absorptive pathway and a chloride-secretory pathway. To increase the yield of respiratory epithelial cells harvested from mice, we have found the nasal septum is a superior source when compared with the trachea. The nasal septum increases the yield of respiratory epithelial cells up to 8-fold.
The development of primary culture models of transgenic mouse tracheal epithelial cells has greatly facilitated the study of human respiratory diseases. These primary culture models are confluent, fully differentiated, ciliated respiratory epithelium at an air-liquid interface on a semipermeable membrane that mimic many characteristics of murine tracheal epithelial cells in vivo. Prior to the development of murine models, suboptimal methods involving non-polarized and sometimes poorly differentiated primary cultures and immortalized cell lines were used for respiratory epithelial cell research. These models made logistical sense because they often supplied copious numbers of cells through expansion and passage of the cells. While the murine air-liquid interface models are ideal for the development of differentiated respiratory epithelial cells, expansion and passage of these cells will ultimately decrease the ability for these cells to differentiate (1). Therefore, one of the primary limitations of in vitro murine models includes the large number of mice required to obtain a significant number of tracheal epithelial cells. Typically, dissociation of respiratory epithelium from two mouse tracheas is required for the development of an air-liquid interface on one transwell membrane (6.5-mm diameter). This can be cost-prohibitive when attempting to develop in vitro airway models of transgenic mice. An additional source of respiratory epithelium will increase the utility of these mice and, at the same time, decrease expenses.
Our recent investigations have found that the nasal septum is a superb source of murine respiratory epithelium. Most research involving murine nasal septa has primarily focused on olfaction, so the potential for respiratory cell culture has largely been ignored. The two sides of the murine nasal septum are covered with mucosa, and the overall surface area is much larger in comparison to the mouse trachea. The olfactory system of the mouse has four anatomically distinct chemosensory areas on the nasal septum. (Figure 1) The main olfactory epithelium (OE), the septal organ of Masera (SO), and the vomero-nasal organ (VNO) have bipolar sensory neurons that reside in a pseudostratified neuroepithelium. The MOE and SO are part of the main olfactory system, which primarily detects odorant molecules, while the VNO detects pheromones and is the major component of the accessory olfactory system (2,3). The fourth anatomically distinct chemosensory area of the nasal septum is the septal organ of Grüneberg (SOG) (4). This chemosensory area is submucosal in location and covered with respiratory epithelium. Approximately 50% of the total surface area of the septum is respiratory epithelium. It has been presumed that nasal respiratory epithelium is very similar to tracheal respiratory epithelium in structure and function. Furthermore, sinonasal disorders mimic or coexist with many respiratory diseases, such as cystic fibrosis, aspirin-sensitive asthma with polyps, and allergic fungal sinusitis (the upper airway correlates to allergic bronchopulmonary aspergillosis) (5). Thus we have focused our efforts on establishing air-liquid interface cultures from the mouse nasal septum and demonstrate that our technique increases the yield of respiratory epithelium 8-fold. Additionally, our technique for removing the nasal septum is simple and straightforward.
Materials and Methods Tissue Culture TechniqueHarvest of mouse nasal septum.
Following euthanasia with a CO2 gas chamber and cervical dislocation, the mouse was placed on a Styrofoam® dissection table in the prone position and secured with several 18-gauge needles. The skin at the nape of the neck was incised with fine dissecting scissors, and the incision rotated around the entire neck. This skin was then dissected anteriorly and completely removed, exposing the bone over the entire skull and nose. The skull was then sectioned in the coronal plane posterior to the eyes (Figure 2A, cut no. 1). The remnant of the brain was completely removed, leaving the anterior aspect of the skull base exposed. The most anterior point of the skull base on either side is directly posterior to the mouse nasal cavities. The remaining portion of the skull was removed to the posterior aspect of the nasal cavity (Figure 2A, cut no. 2).