Yan Ziying, Engelhardt John F, Yuan Feng
Division of Pulmonary, Allergy and Critical Care Medicine, Heersink School of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.
Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA.
Bio Protoc. 2025 Jul 20;15(14):e5399. doi: 10.21769/BioProtoc.5399.
Well-differentiated airway epithelial cultures are commonly used to study airway stem cell lineages, ion and fluid transport, respiratory virus infection and replication, and disease mechanisms in vitro. This culture model involves the isolation and expansion of airway stem cells followed by their differentiation at an air-liquid interface (ALI), a process that has been previously documented in humans and mice. Domestic ferrets () have gained considerable importance in respiratory disease research due to their notable susceptibility to these conditions and their anatomical similarities to humans. Here, we present a comprehensive description of the isolation and culture of stem/progenitor cells from the ferret airway, along with a protocol for their differentiation at the ALI. Our findings have demonstrated that this ferret culture system not only supports the differentiation of the predominant airway epithelial cell types but also facilitates the generation of rare airway epithelial subpopulations, including pulmonary ionocytes, tuft cells, and pulmonary neuroendocrine cells. Additionally, we provide a detailed procedure for measuring transepithelial ion transport relevant to airway diseases, particularly cystic fibrosis. The ability to isolate and culture ferret airway stem cells, combined with ALI differentiation and functional assessment of transepithelial ion transport, offers a powerful platform for evaluating genetic and pharmacologic interventions related to cystic fibrosis. Key features • A protocol for isolating ferret airway basal cells and generating air-liquid interface (ALI) cultures for electrophysiologic research. • Detailed procedures for propagating ferret airway basal cells and culturing in vitro well-differentiated airway epithelium. • A protocol for measuring ion transport, conductance, and immunofluorescence to identify airway cell types.
高分化气道上皮培养物常用于体外研究气道干细胞谱系、离子和液体转运、呼吸道病毒感染与复制以及疾病机制。这种培养模型涉及气道干细胞的分离和扩增，随后使其在气液界面（ALI）分化，这一过程先前已在人类和小鼠中得到记载。由于家雪貂对这些病症具有显著易感性且其解剖结构与人类相似，因此在呼吸道疾病研究中具有相当重要的地位。在此，我们全面描述了从雪貂气道分离和培养干/祖细胞的方法，以及它们在ALI处分化的方案。我们的研究结果表明，这种雪貂培养系统不仅支持主要气道上皮细胞类型的分化，还促进了包括肺离子细胞、簇状细胞和肺神经内分泌细胞在内的罕见气道上皮亚群的生成。此外，我们提供了一种详细的程序，用于测量与气道疾病（尤其是囊性纤维化）相关的跨上皮离子转运。分离和培养雪貂气道干细胞的能力，结合ALI分化和跨上皮离子转运的功能评估，为评估与囊性纤维化相关的基因和药物干预提供了一个强大的平台。关键特性 • 一种分离雪貂气道基底细胞并生成用于电生理研究的气液界面（ALI）培养物的方案。 • 扩增雪貂气道基底细胞并培养体外高分化气道上皮的详细程序。 • 一种测量离子转运、电导和免疫荧光以鉴定气道细胞类型的方案。
