46 vitro culturing of the mucociliary tissue reflects the complexity of the mucociliary differentiation in vivo (see Dimova et al. 2005; Wu et al. 1985). Factors to be considered cover different levels of the culturing procedure, and include: processing of the source tissue or cells, seeding density, confluence and cell differentiation status, type of cellular support used (membrane versus cell culture vessel, ECM molecules used for coating), medium compo- sition, feeding regimen, culture time, and number of passages. None of these factors alone appear indispensable or sufficient to direct the differentiation of respiratory cells in vitro. Among a variety of aspects involved in mucociliary differentiation, the cell-to-cell interactions seem to be one of the most important. In vitro, these interactions depend on several conditions, like the processing of the source tissue, seeding density, the presence of cell support, coating vessels with ECM molecules, and the number of passages. The processing of the source tissue (i.e., inoculating culture with non-dissociated versus dissociated cells) appears to have the most immediate consequences. Undisrupted cell contacts are present in suspension cultures inoculated with undigested mucosal sheets (collected by nasal brushing). Mucociliary phenotype in these cultures directly reflects the differentiation status of the source tissue. In contrast, in cultures inoculated with dissociated cells, high seeding density is required for the promotion of cell proliferation and adhesion, which accelerates cellular confluence (Neugebauer et al. 2003; Kowalski et al. 1998). Once the culture reaches confluence, epithelial cells are forced to change shape, increasing contact with their neighbors. This results in a better transduction of the differentiation signals, which prompt polarization of the cell layer, formation of tight junctions on the lateral sides of the cells, and, finally, the growth of microvilli and cilia on the apical cell surfaces (Neugebauer et al. 2003). However, the successful cell differentiation can proceed in vitro only in the presence of medium containing a balanced composition of factors that facilitate both cellular proliferation and differentiation. The intact network of cell-to-cell interactions together with the proper medium composition are sometimes sufficient to maintain or induce cell differentiation, as evidenced by the success of suspension cultures initiated from mucosal sheets (Bridges et al. 1991b; Neugebauer et al. 2003). In cultures initiated from dissociated cells, the development of cell-to-cell interactions is required for proper polarization of the cell layer and for the further differentiation process. Following the initial orientation of the apical and basal surfaces of the cells, further steps of the differentiation occur, ending in the formation of typical secretory or columnar (ciliated or non-ciliated) RE cells (Crystal et al. 2008). The polarization and differentiation J Appl Genetics (2011) 52:39–51 processes can be promoted by additional external signals, such as a specific type of culture (suspension or ALI culture described above), special cell support (culture matrix or vessel coating), or the composition of medium supplements (reviewed in Schmidt et al. 1998; Dimova et al. 2005). ECM molecules, constituents of the basement mem- brane in RE, are naturally involved in the RE cell proliferation, migration, and differentiation. They are also essential for the development of a successful culture of human RE cells. Different ECM molecules have been used for that purpose (Table 4), but collagen seems to be the most promising. Coating of the culture vessels with collagen (especially type I), as opposed to laminin, fibronectin, and polylysine (Table 4), has been reported to positively influence attachment, growth (Wu et al. 1985), and differentiation (Neugebauer et al. 2003) of the RE cells in culture. In submerged cultures, the effect of collagen coating depends on its physical structure. RE cells grown on a derivatized collagen showed better differentiated phenotype (monolayer with columnar/cuboidal morphology), com- pared to cells grown on a fibrillar or polymerized collagen, which promoted squamous and multilayered phenotype (Agu et al. 2001). In cultures grown on porous membranes (submerged and ALI), the effects of collagen coating are not so obvious/straightforward. Some investigators reported the improvement of cell attachment efficiency, cell prolif- eration, and differentiation (Yankaskas et al. 1985; Clark et al. 1995), while others reported no influence (Werner and Kissel 1995) or even negative effects on cell growth (Wu et al. 1985). These contradictory results suggest that other factors, such as media composition and possibly the source of the cells, are important for the full expression of the ECM influence. Media composition strongly influences the viability, proliferation, and differentiation of any cultured cell type, including RE cells. In the absence of a proper medium, efficient mucociliary differentiation is not achieved even in, otherwise most favorable, ALI cultures. Conversely, a proper medium composition can be sufficient to induce ciliogenesis even in cultures grown in suspension or in the absence of coating with ECM molecules (Neugebauer et al. 2003) (Table 5). Successful in vitro ciliogenesis/differentia- tion can occur in both simple (only 2–3 supplements) (Werner and Kissel 1995; Agu et al. 2001) and more complicated media (Wu et al. 1985) (Table 5). For a detailed review of different media components and their influence on cell growth and differentiation, see Dimova et al. (2005). Already the early studies had shown that serum- supplemented media, most suitable for the culture of bronchial cells, would limit the cell proliferation and lifespan of tracheal and nasal epithelial cells (Masui et al. 1986). Serum was also shown to induce squamous cell