44 atraumatic sampling described in the literature (Schmidt et al. 1998), only nasal scraping (using a small curette) and brushing (using a small cytobrush) give the yield of epithelial cells high enough to start a primary cell culture (Bridges et al. 1991b; Lopez-Souza et al. 2003). Specimens collected by these methods usually include only the epithelium, without the deeper layers of the mucosa. Importantly, atraumatic methods provide nasal epithelium, without the ion transport abnormalities typical for nasal polyps (Bernstein and Yankaskas 1994; Schmidt et al. 1998). Mucociliary phenotype in the in vitro cultures of RE cells The primary goal of in vitro cell culture systems is to achieve differentiated morphology and biochemical fea- tures, resembling original tissue as closely as possible (Dimova et al. 2005). This can mean maintaining the differentiated state of the source cells or reconstituting the differentiated state in the cells following their in vitro proliferation; the latter is especially important if the culture is to be used in the differential diagnosis of PCD and SCD. The history of development of culturing methods that would allow achieving the expression of typical functions of the RE, such as barrier formation, metabolic capacity, vectorial transport of solutes, as well as mucus production and ciliary activity, shows that it was not an easy task (Dimova et al. 2005). One of the important issues to start with was that lifespan of differentiated cells in cultures of nasal RE cells which in the beginning was only a few days. Initial culture conditions were optimized for cell attachment, proliferation, and further subculturing. As a consequence, freshly seeded mucociliary-differentiated cells would lose their cilia and secretory granules within a few days (Bernacki et al. 1999). At the same time, newly proliferated cells followed the pathway of squamous differentiation, which is induced in vivo under conditions of chemical or mechanical injury or vitamin A deprivation (Rearick and Jetten 1989). In effect, a stratified epithelium was formed, with the top layer of squamous, flattened cells and without any ciliated or secretory cells (Rearick and Jetten 1989). The retention of the mucociliary phenotype lasted slightly longer in explant outgrowth cultures (see below), presumably due to the supportive influence of non-epithelial cell types (Agu et al. 1999; Dimova et al. 2005; Neugebauer et al. 2003). With time, conditions of long-term cultures were established, supporting both cell proliferation and muco- ciliary differentiation of newly formed cells, together with the retention of the pseudostratified phenotype. A success in mucociliary differentiation was achieved using a number of different methods (Chevillard et al. 1991, 1993; de Jong et al. 1994; Neugebauer et al. 2003; Jorissen et al. 1989). The direction and extent of the differentiation depended on the culturing system and many other factors described below. J Appl Genetics (2011) 52:39–51 Culture systems There are two basic approaches to establishing the culture of differentiated RE cells: explant growth cultures and cultures of dissociated tissues (Table 3). Explant outgrowth cultures were one of the first systems developed. Small specimens of tissue (biopsies) were cultured on uncoated (Steele and Arnold 1985) or coated plastic supports (collagen, ECM molecules, etc.) (Wiesel et al. 1983). Sometimes, also a fibroblast feeder layer was used (de Jong et al. 1993). The most promising aspect of this system was its high reproducibility. Explants could be serially replated up to seven times and, after the removal of the explant, cultures retained ciliary activity (Wiesel et al. 1983). Although high levels of cell differentiation were achieved (Chevillard et al. 1991), the results of the experiments were not easy to predict and to interpret, due to the presence of non-epithelial cell types in explants. Another disadvantage was the longer time needed to establish the cell culture, compared with the cultures starting from dissociated cells (Dimova et al. 2005). The use of explant outgrowth cultures became, therefore, less popular; presently, this approach is most often used in studies on the immune response to pathogens and allergens (Ooi et al. 2007; Liu et al. 2007), and on the epithelial barrier function in allergic rhinitis. Another effective approach to culturing differentiated RE cells is based on use of dissociated tissue samples (Table 3). Digestion of a sample with a protease (pronase) at a low temperature has proven to be one of the most efficient ways to isolate a pure population of epithelial cells, and dispose of non-epithelial cells (Dimova et al. 2005; Schmidt et al. 1998; Wu et al. 1985). Dissociated cells also allow a faster establishment of the culture (Dimova et al. 2005). The system underwent a long evolution since the first reports in the 1980s. The conditions of pronase digestion were optimized, together with other techniques reducing the fibroblast contamination (serum-free media, preplating of the dissociated cells mixture on plastic supports), and cell cluster formation (filtering the cell suspension through filters or sieves) (reviewed in Dimova et al. 2005). Of note, in contrast to solid-tissue samples, samples collected by nasal Table 3 Culture systems Explant growth Dissociated tissue Adherent: Submersion Air–liquid interface (ALI) Suspension Sequential: Submersion monolayer followed by suspension