Long-Term Cultures of Polarized Airway Epithelial Cells from Patients with Cystic Fibrosis

Abstract

The poor ability of respiratory epithelial cells to proliferate and differentiate in vitro into a pseudostratified mucociliated epithelium limits the general use of primary airway epithelial cell (AEC) cultures generated from patients with rare diseases, such as cystic fibrosis (CF). Here, we describe a procedure to amplify AEC isolated from nasal polyps and generate long-term cultures of the respiratory epithelium. AEC were seeded onto microporous permeable supports that carried on their undersurface a preformed feeder layer of primary human airway fibroblasts. The use of fibroblast feeder layers strongly stimulated the proliferation of epithelial cells, allowing the expansion of the cell pool with successive passages. AEC at increasing passage were seeded onto supports undercoated with airway fibroblasts and exposed to air. Either freshly isolated or amplified AEC could differentiate into a pseudostratified mucociliated epithelium for at least 10 mo. Thus, CF epithelia cultures showed elevated Na+ transport, drastic hyperabsorption of surface liquid, and absence of cAMP-induced Cl- secretion as compared with non-CF cultures. They were also characterized by thick apical secretion that hampered the movement of cell surface debris by cilia. However, CF respiratory epithelia did not show increased production of mucins or IL-8. The method described here is now routinely used in our laboratory to establish long-term cultures of well differentiated respiratory epithelia from human airway biopsies.

Profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways

“…Because of the crucial role of this anabolic factor in the induction of cell survival and gene neotranscription (26), this result further supports the concept that neutrophils, upon recruitment to CF airways, modify their default program to survive and undergo potential functional changes, likely caused by conditioning by CF epithelial cells (30,41). The maintenance of high levels of phosphorylated Akt, which has strong anti-apoptotic and anabolic activities (42), is consistent with an increased survival of CF airway neutrophils.…”

Approaches to Study Differentiation and Repair of Human Airway Epithelial Cells

“…Two days after seeding, hAECs are switched to an air-liquid interface for at least 45 days. This leads to the differentiation of hAECs to a mucociliated pseudostratified airway epithelium that is maintained in a homeostatic state for months (11). The transparent microporous membrane allows direct observation of the cells under a conventional inverted microscope.…”

“…The culture is usually viable for about 1 month after the beginning of the culture (4). The other model consists in a first step of hAEC proliferation followed by a second step of differentiation (10,11). These models present the advantage to amplifying a rare material, especially for CF tissues.…”

Hyperinflammation in airways of cystic fibrosis patients: what’s new?

“…The airway epithelium is an important source of chemokine IL-8 and is the principal neutrophil chemoattractant in the CF lung [16]. During the past decade, cell culture systems using primary human bronchial epithelial cells and respiratory cell lines have revealed that CF cells exhibit enhanced proinflammatory signaling compared with non-CF cells [17][18][19][20][21][22][23]. This intrinsic inflammation was mainly characterized by an increased level of inflammatory mediators (e.g., IL-8 and IL-6) compared with non-CF bronchial epithelial cells, either in the absence [19,21,24] or presence of bacterial stimulation [25][26][27][28].…”