Introduction: Emerging as one of the major causes of disability and death, respiratory diseases affect millions of people worldwide.
To date, despite numerous efforts and clinical attempts, there are no effective treatments to repair long-segment defects, and a standard procedure is still missing.
Among the ventured clinical applications, Tissue Engineering (TE) approaches hold the potential to address the main barriers related to surgical-based procedures, which were all negatively affected by the absence of an integer functional epithelium. A self-renewing well-differentiated epithelium is instrumental for all the physiological functions of the respiratory system, avoiding infections and granulation tissue formation.
In TE strategies, autologous adult airway epithelial cells would be the most appropriate cellular source for restoring the airway epithelium. However, many difficulties have been met during their expansion, and these cells were described as effectively able to divide for a very limited number of passages before losing their differentiative potential, thus being considered an unsuitable cell source for TE approaches.
In the present study, we tested the ability of a culture system, largely used for the clinical expansion of different epithelial tissues, to safely and effectively maintain the long-term proliferative and differentiative potential of airway epithelial cells. Moreover, we established quality controls to be adopted in each step aimed at developing a successful TE construct.
Methodology. Human primary epithelial cells derived from the trachea and the bronchi of different donors were isolated and expanded through a clinical-grade culture condition. During the expansion process, reproducible quality controls were set up to verify i) the quality of the cells extracted from the biopsy in terms of tissue-regenerative properties, ii) the maintenance of the cellular identity, iii) the expression of specific markers for the identification of the different cell types, and iv) the tissue integrity, while single cells assays allowed the identification of stem cells within the cultures.
Finally, to obtain a terminally differentiated epithelium able to mimic the in vivo condition, airlifted cultured were developed.
Results. Epithelial cells extracted from human tracheal and bronchial biopsies were effectively cultured for several passages until replicative senescence, proving the capability of the adopted cultured system to sustain the proliferation of airway epithelial cells. Systematic characterization of cultures during serial passages highlighted the maintenance of differentiative potential throughout the expansion process, expressed as the ability of epithelial cells to differentiate into the main airway cellular types, while the expression of tissue-integrity markers attested the reconstruction of an integer epithelium.
Moreover, the establishment of an air-lifted culture mimicking the in vivo condition allowed to obtain a terminally differentiated airway epithelium expressing also ciliated cells.
Finally, single-cell assays and stem-proliferative markers expression detected the presence of stem cells within the cultures, mandatory for any long-term resolutive TE therapy aimed at restoring the airway district.
Conclusion. The proposed clinically-validated culture system succeeded in establishing a safe and effective expansion of airway epithelial stem cells, allowing the set-up of quality controls and paving the way towards the development of future successful TE applications.