Department of Clinical Research, University of Bern, Bern, Switzerland.
Division of Respiratory Medicine, University Children's Hospital of Bern, Bern, Switzerland.
Respir Res. 2017 Dec 28;18(1):215. doi: 10.1186/s12931-017-0706-7.
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. 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.
Differentiated 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.
Pediatric 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.
In 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.
原代囊性纤维化 (CF) 气道上皮细胞的体外系统是研究天然呼吸道上皮分子和功能特征的重要工具。然而，在浸没条件下培养的未分化 CF 气道细胞培养物不能恰当地代表生理情况。基于在气液界面 (ALI) 生长的气道上皮细胞的更先进 CF 细胞培养系统可以再现大多数类似体内的特性，但需要使用侵入性采样方法。在这项研究中，我们描述了通过对儿科患者进行非侵入性鼻刷获得的完全分化的原代 CF 气道上皮细胞的详细特征。
通过对纤毛、分泌黏液和基底细胞以及紧密连接和 CFTR 蛋白的免疫标记来评估分化细胞培养物。通过组织学和透射电子显微镜检查上皮形态和超微结构。通过视频显微镜方法研究纤毛摆动频率，并使用上皮伏特欧姆计系统评估跨上皮电阻。最后，通过细胞层完整性测试和酶联免疫吸附测定法测量基础细胞因子水平来分析上皮通透性。
在 ALI 上生长的儿科 CF 鼻培养物显示出向具有黏液纤毛表型的假复层上皮分化。免疫荧光分析还显示存在纤毛、分泌黏液和基底细胞以及紧密连接。CFTR 蛋白表达在 CF (F508del/F508del) 和健康培养物中均有观察到，并且在 CF 和对照 ALI 培养物中，基础白细胞介素 (IL)-8 和 IL-6 释放相似。纤毛摆动频率为 9.67 Hz，分化的儿科 CF 上皮被发现具有功能紧密性。
总之，在 ALI 上生长的原代儿科 CF 鼻上皮细胞培养物完全分化为纤毛、分泌黏液和基底细胞，充分反映了人类呼吸道上皮的体内特性。
