Table 5 Examples of methods successfully used for culturing differentiated epithelial respiratory cells Neugebauer et al. 2003 Agu et al. 2001 Jorissen et al. 1991 Submerged Inferior turbinates Suspension ALI Sequential Inferior turbinates Nasal polyps or Nasal polyps turbinates 0.01% pronase, 18–22 0.2% pronase, 16–20 0.1% pronase, h at 4°C h at 4°C 16–24 h at 4°C Uncoated plastic Cell vessels uncoated or 0.2% rat tail collagen flasks coated with different gel forms of collagen I J Appl Genetics (2011) 52:39–51 Author Culture type Tissue source Wiesel et al. 1983 Explant outgrowth Nasal polyps Dissociation Mechanical 0.01% pronase, 18– fragmentation 22 h at 4°C Support Endothelial cell- Plastic dishes coated produced with mixture of ECM matrix collagen IV: laminin:heparan sulfate proteoglycan (5:2:2) Seeding 5–7 explants per density 35-mm dish Medium and RPMI 1640, DMEM/F12 (3:1), supplements 25% FCS INS (1 μg/ml), TR (1 μg/ml), HC (0.5 μg/ml), EGF (10 ng/ml), RA (10 ng/ml), L- glutamine (3.2 mM), and 1.25% (v/v) AA Culture time 14 days 5–6 weeks, up to 14 weeks Differentiation Ciliary activity, Densely packed, status cells cuboidal, cuboidal cells with flat, closely small apical packed surface, microvilli and/or cilia Bridges et al. 1991a, b Suspension Bilateral nasal brushing No dissociation 47 DMEM/F12 (3:1), INS (1 μg/ml), TR (1 μg/ml), HC (0.5 μg/ml), EGF (10 ng/ml), RA (10 ng/ml), L- glutamine (3.2 mM), and 1.25% (v/v) AA At least 7 weeks Cells columnar/ cuboidal, densely packed, bearing cilia and/or microvilli 106 cells/cm2 First day: DMEM/F12 (1:1), 5% FCS Later: DMEM/F12 (1:1), 2% UltroSer G, CT (10 ng/ml), strep (50 μg/ml), pen (50 ng/ml) 12 days Derivatized collagen I: monolayer with columnar/cuboidal ciliated and non- ciliated cells Less than 103 cells/cm 2 Monolayer: DMEM/ F12 (1:1), 2% UltroSer G, pen (50 μg/ml), strep (50 mg/ml) Suspension: DMEM/ F12 (1:1), 10% NuSerum, pen (50 μg/ ml), strep (50 mg/ml) Up to 28 weeks In suspension, cells cuboidal to columnar, microvilli and cilia, no goblet cells visible F12 with INS (2 μg/ ml), HC (100 nM), CT (10 ng/ ml), T3 (2 nM), EGCS (4 ug/ml), EGF (12.5 ng/ml) Up to 14 days Polarized cells: microvilli/cilia visible DMEM, Dulbecco’s Modified Eagle’s Medium; F12, Ham’s F12 Medium; INS, insulin; TR, transferrin; HC, hydrocortisone, EGF, epithelial growth factor; RA, retinoic acid; CT, cholera toxin; pen, penicillin; strep, streptomycin; AA, antibiotic–antimycotic solution differentiation and to impair ion transport in respiratory epithelial cells (Van Scott et al. 1988). This inhibition was later shown to be due to the presence of a transforming growth factor β in the blood-derived serum (Masui et al. 1986). With time, the composition of serum-free hormone- supplemented media was optimized to enhance growth and to prolong the lifespan of cultured respiratory epithelial cells (Lechner and LaVeck 1985; Wu et al. 1985). Serum replacements such as UltroSer G, formulated to support the growth of airway epithelial cells, are now commercially available. The most frequently used serum-free media are: DMEM, F12, and BEGM (Agu et al. 2001; Dimova et al. 2005; Jorissen et al. 1989; Schmidt et al. 1998) (Table 5), and, although rather seldom, sometimes also, RPMI 1640 was used (Wiesel et al. 1983) (Table 5). The media are often complemented with antibiotics and fungicides (e.g., penicillin, streptomycin, gentamicin, amphotericin B), in order to reduce microbial and yeast contamination, which is typical for the tissues exposed to the external environment (Jorissen and Willems 2000; Werner and Kissel 1995; Yankaskas et al. 1985; Yoon et al. 2000) (Table 5). A broad range of supplements can be added to the culture media to promote better attachment, growth, and differentia- tion of RE cells: insulin, hydrocortisone, epithelial growth factor (EGF), epinephrine, triiodothyronine, bovine pituitary extract, endothelial cell growth supplement, transferring ethanolamine, phosphoethanolamine, retinoic acid and its derivatives, and cholera toxin (Bridges et al. 1991b; Jorissen et al. 1989; Lechner and LaVeck 1985; Sachs et al. 2003; Werner and Kissel 1995; Yankaskas et al. 1985) (Table 5). Insulin is considered to be the most important supplement promoting cell growth (Gray et al. 1996; Lechner and LaVeck 1985; Wu et al. 1986), but for the majority of media components, the physiological importance is either not yet precisely defined or depends on the culture method used. Sometimes, the components are added just in order to follow previously described methods which have been optimized experimentally in cells from other species. As a conse- quence, the composition of culture media and supplements is very variable and, in many cases, gives contradictory results. The action of some supplements may depend on the presence and concentration of other media components.