48 Retinol is considered to be an important factor in maintaining the proliferation and differentiation of RE cultures. Its action is highly dependent on the calcium level (Sachs et al. 2003; Wu et al. 1985) and on specific cellular support (Rearick and Jetten 1989). On the other hand, high calcium level combined with high EGF level is known to suppress mucociliary differentiation and promote the transition of cultured human respiratory cells towards squamous epithelium (Sachs et al. 2003; Van Scott et al. 1988). Therefore, for in vitro mucociliary differentiation of the human respiratory epithelial cells, it is suggested to use media containing a high concentration of Ca ions (∼1 mM), high retinoic acid, and low EGF (Sachs et al. 2003). In addition, it is recommended to use collagen matrices (coating, gel, membranes), as they help to express the secretory phenotype in retinoic acid containing cultures (Rearick and Jetten 1989). The effects of many supplements are not consistent or well-pronounced; their use can depend on the cell origin (species), culture method used, and the scientific goal of the study. For example, insulin, cholera toxin, and bovine pituitary extract were considered to be essential for cell growth in some cases (Wu et al. 1985), while in other settings, sustained cell proliferation was observed only if insulin, EGF, hydrocortisone, ethanolamine, and phosphoe- thanolamine were present (Lechner and LaVeck 1985). In addition, the optimal concentrations of supplements can differ depending on a specific type of culture; for example, it has been observed that EGF concentrations required for growth and mucociliary differentiation in ALI cultures are much lower compared to those in submerged cell culture type (Schmidt et al. 1998; Dimova et al. 2005). Conclusions Differentiated cultures of the respiratory epithelium (RE) cells offer an important augmentation of the experimental toolbox in the diagnosis and analysis of the molecular basis of primary ciliary dyskinesia (PCD). Due to the process of de novo mucociliary differentiation in the absence of harmful environmental factors, the cultures permit the exclusion of any acquired changes in the ciliary architecture and, in consequence, the reliable differential diagnosis of PCD versus secondary ciliary dyskinesia (SCD). By providing the tissue required for the simultaneous analysis of the ciliary ultrastructure and function, the cultures allow deeper characterization of the genotype–phenotype relation- ships in the ciliary protein mutants. Several types of successful culture systems of differen- tiated RE cells have been reported to date, and each of them has its advantages and disadvantages. The existing systems are generally no superior to each other, and still require J Appl Genetics (2011) 52:39–51 better definition and standardization. Still, a specific cell culture system has to be selected depending on the specific application or the scientific goal of the study. Although differentiated cultures of respiratory epithelial cells permit the diagnosis of PCD and/or SCD, the same cultures are not always suitable for the simultaneous research on transport, metabolism, toxicity, and mucociliary differentiation. Therefore, the evolution of a perfect in vitro cell system that would allow separate studying of all of the important processes and cell types of the airway epithelium still remains a goal for the future. Acknowledgments This study was supported by the Polish Ministry of Science grant number NN401-09-5537 and the EU grant number ECFP7-HEALTHPROT-GA229676 (ZB). The authors declare that they have no conflict of interest. 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