J Appl Genetics (2011) 52:39–51 scraping or brushing techniques do not require additional digestion. The collected cells are already present in the form of mucosal sheets, single cells, and cell clusters, similarly to tissue samples which were digested with protease but not sieve-filtered (Bridges et al. 1991b; Neugebauer et al. 2003). There are many types of cultures in which the dissociated RE cells are used (Table 3). They include adherent culture (which can be further divided into submersion and air–liquid interface [ALI]), suspension culture, and sequential submer- sion monolayer-suspension culture (Gray et al. 1996; de Jong et al. 1994; Jorissen et al. 1989; Bridges et al. 1991b). In the adherent cultures, dissociated epithelial cells were originally seeded in plastic uncoated vessels (Gray et al. 1996) or on a layer of fibroblast feeder cells (Claass et al. 1991; de Jong et al. 1993; Wu et al. 1985). Later, it was found that the adhesion and proliferation of epithelial cells were better promoted by the use of different growth supports/matrix (plastic dishes, floating gel, semipermeable membranes), and vessel/membrane coating with ECM molecules (collagen type I, laminin, etc.), better mimicking the composition of basement membrane, which supports the RE in the in vivo setting (Neugebauer et al. 2003) (Table 4). Finally, the submersion method used in most of the adherent cultures was found to have inhibitory effects on the process of mucociliary differentiation; differentiated cells were losing cilia and proliferated cells remained undifferentiated (Bernacki et al. 1999), probably due to the lack of signals such as air interface or contact with ECM molecules, which induce cell polarization. A better degree of differentiation was obtained in suspension cultures (Table 3). When epithelial cells collected by nasal brushing were cultured in the form of polarized multicellular spheroids, with only minimal adher- ence of epithelial cells to the support, cells retained the ciliated phenotype for over 14 days (Bridges et al. 1991b). After a longer culture time (21 days) in a similar setting (nasal brushing without cell dissociation), the de novo formation of cilia could be observed (Pifferi et al. 2009). An important modification of the adherent cultures is the use of the ALI (Table 3). In this method, adherent cells Table 4 Cell supports, mem- branes, and coatings used in cultures of differentiated respiratory epithelium (RE) cells Cell support material Uncoated plastic Membranes Polyester Polyethylene terephthalate Hydrophilic polytetrafluoroethylene Polycarbonate Mixed cellulose esters Collagen membranes CD-24 Hyaluronate derivatives 45 growing on inserts with porous bottoms are fed only from their basolateral side, and their apical surface has contact with the air, reflecting the in vivo situation in the RE (de Jong et al. 1993; Wu et al. 1986). Such polarization of the cell layer allows much higher levels of differentiation than a typical submersion feeding system (Whitcutt et al. 1988; Kondo et al. 1991, 1993). The ALI is now a widely recognized “gold standard” for culturing RE cells. Due to the presence of a permeable insert supporting adherent cells, ALI systems are especially useful for measuring the transport and metabolism of drugs across the cell layer and within the cells (Schmidt et al. 1998; Dimova et al. 2005). The drawback of this method is a rather short time for which the layer of differentiated cells is available: in some cases, cells started to detach from the culture vessels already ∼14 days after reaching conflu- ence (Werner and Kissel 1995). The long life of a differentiated culture is the main advantage of the sequential monolayer-suspension culture method (Jorissen and Bessems 1995) (Table 3). During the first phase, the source cells are cultured as an adherent layer in culture vessels coated with collagen type I gel. When cells reach confluency (the time depends on the seeding density), the monolayer is detached from the vessel by use of a collagenase, mechanically fragmented, and transferred into the suspension culture. During the next week, suspended cells are mechanically rotated, in order to promote the formation of closed spheroids from monolayer fragments. Cells in the spheroids are well differentiated and cilia are developed at this stage. Then, ciliated spheroids can be cultured in a stationary incubator, for up to 7 months (Jorissen et al. 1989). The main disadvantage of the sequential method is that it is time-consuming and rather costly. Factors influencing differentiation in vitro Mucociliary differentiation has been demonstrated in many different culture systems, but it remains a difficult task, with only a few laboratories having achieved success in this field (Bridges et al. 1991b; Chevillard et al. 1993). The broad range of conditions reported for the successful in Coating Collagen type I Rat tail collagen Calf-skin collagen Bovine dermal collagen (Vitrogen 100) Bovine placental collagen Collagen type IV Human placental collagen Mixtures: Collagen type IV:laminin:heparan sulfate proteoglycan