Characterization of pediatric cystic fibrosis airway epithelial cell cultures at the air-liquid interface obtained by non-invasive nasal cytology brush sampling

Aline Schögler,Seraina Beyeler,Melanie Brügger,Nicolas Regamey,Marco P Alves,Thomas Geiser,Fabian Blank,Stefan A Tschanz,Carmen Casaulta

doi:10.1186/s12931-017-0706-7

Abstract

BackgroundIn vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. However, undifferentiated CF airway cell cultures grown under submerged conditions do not appropriately represent the physiological situation. A more advanced CF cell culture system based on airway epithelial cells grown at the air-liquid interface (ALI) recapitulates most of the in vivo-like properties but requires the use of invasive sampling methods. In this study, we describe a detailed characterization of fully differentiated primary CF airway epithelial cells obtained by non-invasive nasal brushing of pediatric patients.MethodsDifferentiated cell cultures were evaluated with immunolabelling of markers for ciliated, mucus-secreting and basal cells, and tight junction and CFTR proteins. Epithelial morphology and ultrastructure was examined by histology and transmission electron microscopy. Ciliary beat frequency was investigated by a video-microscopy approach and trans-epithelial electrical resistance was assessed with an epithelial Volt-Ohm meter system. Finally, epithelial permeability was analysed by using a cell layer integrity test and baseline cytokine levels where measured by an enzyme-linked immunosorbent assay.ResultsPediatric CF nasal cultures grown at the ALI showed a differentiation into a pseudostratified epithelium with a mucociliary phenotype. Also, immunofluorescence analysis revealed the presence of ciliated, mucus-secreting and basal cells and tight junctions. CFTR protein expression was observed in CF (F508del/F508del) and healthy cultures and baseline interleukin (IL)-8 and IL-6 release were similar in control and CF ALI cultures. The ciliary beat frequency was 9.67 Hz and the differentiated pediatric CF epithelium was found to be functionally tight.ConclusionIn summary, primary pediatric CF nasal epithelial cell cultures grown at the ALI showed full differentiation into ciliated, mucus-producing and basal cells, which adequately reflect the in vivo properties of the human respiratory epithelium.

Highlights

In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium
In a more advanced in vitro cell culture model, airway epithelial cells are grown at the air-liquid interface (ALI) and re-differentiated into a ciliated pseudostratified epithelium covered by a layer of mucus
Whereas ALI cultures based on healthy airway epithelial cells are widely used, a limited number of investigators are working with cell cultures grown at the ALI in the field of CF research, and these fully differentiated CF airway epithelial cell cultures have so far been only partially characterized [4,5,6]

Summary

Introduction

In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. In vitro models based on primary airway epithelial cells are important tools to study molecular and functional aspects of the human respiratory epithelium. In a more advanced in vitro cell culture model, airway epithelial cells are grown at the air-liquid interface (ALI) and re-differentiated into a ciliated pseudostratified epithelium covered by a layer of mucus. This system of a differentiated mucociliary epithelium could serve as a preferable cell culture model that resembles more closely the in vivo properties of the native respiratory epithelium in structure (highly motile cilia and tight junction formation) and function (transepithelial electrical resistance and mucus production/secretion/transport) [3]. Since it is ethically and technically easier to isolate primary nasal compared to bronchial epithelial cells, there is an interest in using nasal cultures as a surrogate of bronchial cells for studies based on airway epithelial cells [11,12,13,14]

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