Optimisation of growth conditions for ovine airway epithelial cell differentiation at an air-liquid interface.
Optimisation of growth conditions for ovine airway epithelial cell differentiation at an air-liquid interface.
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DOI：
10.1371/journal.pone.0193998
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Davies RL
中科院分区：
文献类型：
--
作者：
O'Boyle N;Sutherland E;Berry CC;Davies RL
呼吸道感染是农业中的重要问题。有一个良好的特点，在体外上皮细胞培养模型的发展的需求，以解剖在气道上皮细胞中的宿主-病原体界面发生的各种分子相互作用。我们分析了影响绵羊气管上皮细胞在气-液界面（ALI）培养系统中生长和分化的关键因素。在ALI后21天评估细胞分化，这是我们先前已经证明在标准生长条件下足以分化的时间点。我们确定了剂量依赖性反应表皮生长因子（EGF）的上皮增厚和纤毛水平。最大的纤毛水平观察到25 ng ml-1的EGF。我们确定了一个严格的要求，视黄酸（RA）在上皮细胞分化的RA排除导致形成的复层鳞状上皮，没有纤毛。生长基质的孔密度也对分化有影响，因为高孔密度插入物比低孔密度插入物产生更高水平的纤毛化和更均匀的细胞层。通过在低于环境氧浓度的气氛中培养细胞，分化也得到改善。我们比较了两种浸没式生长培养基，并观察到增殖/扩增速率、屏障形成以及终末分化的差异。两者合计，这些结果表明绵羊气管上皮细胞和其他先前描述的气道上皮模型，各种环境条件下的反应之间的重要差异。这些数据还表明，绵羊气管上皮细胞的表型可以通过精确调节生长条件在体外定制，从而产生可定制的潜在感染模型。
Respiratory tract infections are of significant concern in the agriculture industry. There is a requirement for the development of well-characterised in vitro epithelial cell culture models in order to dissect the diverse molecular interactions occurring at the host-pathogen interface in airway epithelia. We have analysed key factors that influence growth and differentiation of ovine tracheal epithelial cells in an air-liquid interface (ALI) culture system. Cellular differentiation was assessed at 21 days post-ALI, a time-point which we have previously shown to be sufficient for differentiation in standard growth conditions. We identified a dose-dependent response to epidermal growth factor (EGF) in terms of both epithelial thickening and ciliation levels. Maximal ciliation levels were observed with 25 ng ml-1 EGF. We identified a strict requirement for retinoic acid (RA) in epithelial differentiation as RA exclusion resulted in the formation of a stratified squamous epithelium, devoid of cilia. The pore-density of the growth substrate also had an influence on differentiation as high pore-density inserts yielded higher levels of ciliation and more uniform cell layers than low pore-density inserts. Differentiation was also improved by culturing the cells in an atmosphere of sub-ambient oxygen concentration. We compared two submerged growth media and observed differences in the rate of proliferation/expansion, barrier formation and also in terminal differentiation. Taken together, these results indicate important differences between the response of ovine tracheal epithelial cells and other previously described airway epithelial models, to a variety of environmental conditions. These data also indicate that the phenotype of ovine tracheal epithelial cells can be tailored in vitro by precise modulation of growth conditions, thereby yielding a customisable, potential infection model.
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