Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, The Netherlands.
Sci Rep. 2020 Aug 3;10(1):13042. doi: 10.1038/s41598-020-69982-0.
Pulmonary infection is associated with inflammation and damage to the bronchial epithelium characterized by an increase in the release of inflammatory factors and a decrease in airway barrier function. Our objective is to optimize a method for the isolation and culture of primary bronchial epithelial cells (PBECs) and to provide an ex vivo model to study mechanisms of epithelial airway inflammation. PBECs were isolated and cultured from the airways of calves in a submerged cell culture and liquid-liquid interface system. A higher yield and cell viability were obtained after stripping the epithelium from the bronchial section compared to cutting the bronchial section in smaller pieces prior to digestion. Mannheimia haemolytica and lipopolysaccharide (LPS) as stimulants increased inflammatory responses (IL-8, IL-6 and TNF-α release), possibly, by the activation of "TLR-mediated MAPKs and NF-κB" signaling. Furthermore, M. haemolytica and LPS disrupted the bronchial epithelial layer as observed by a decreased transepithelial electrical resistance and zonula occludens-1 and E-cadherin expression. An optimized isolation and culture method for calf PBECs was developed, which cooperated with animal use Replacement, Reduction and Refinement (3R's) principle, and can also contribute to the increased knowledge and development of effective therapies for other animal and humans (childhood) respiratory diseases.
肺部感染与支气管上皮的炎症和损伤有关，其特征是炎症因子的释放增加和气道屏障功能下降。我们的目标是优化一种分离和培养原代支气管上皮细胞（PBECs）的方法，并提供一个体外模型来研究上皮气道炎症的机制。采用淹没细胞培养和液-液界面系统从小牛气道中分离和培养 PBECs。与在消化前将支气管段切成较小的块相比，从支气管段中剥离上皮可获得更高的产量和细胞活力。溶血曼海姆菌和脂多糖（LPS）作为刺激物增加了炎症反应（IL-8、IL-6 和 TNF-α 的释放），可能是通过激活“TLR 介导的 MAPKs 和 NF-κB”信号通路。此外，溶血曼海姆菌和 LPS 破坏了支气管上皮层，表现为跨上皮电阻降低和封闭蛋白-1 和 E-钙黏蛋白表达减少。开发了一种优化的小牛 PBECs 分离和培养方法，该方法符合动物使用替代、减少和优化（3R）原则，也有助于增加对其他动物和人类（儿童）呼吸道疾病的有效治疗方法的了解和发展。
