PDF(Pubmed)
Abstract：
The epithelial lining of the lung is often the first point of interaction between the host and inhaled pathogens, allergens and medications. Epithelial cells are therefore the main focus of studies which aim to shed light on host-pathogen interactions, to dissect the mechanisms of local host immunity and study toxicology. If these studies are not to be conducted exclusively in vivo, it is imperative that in vitro models are developed with a high in vitro- in vivo correlation. We describe here a co-culture model of the bovine alveolus, designed to overcome some of the limitations encountered with mono-culture and live animal models. Our system includes bovine pulmonary arterial endothelial cells (BPAECs) seeded onto a permeable membrane in 24 well Transwell format. The BPAECs are overlaid with immortalised bovine alveolar type II epithelial cells and cultured at air-liquid interface for 14 days before use; in our case to study host-mycobacterial interactions. Characterisation of novel cell lines and the co-culture model have provided compelling evidence that immortalised bovine alveolar type II cells are an authentic substitute for primary alveolar type II cells and their co-culture with BPAECs provides a physiologically relevant in vitro model of the bovine alveolus. The co-culture model may be used to study dynamic intracellular and extracellular host-pathogen interactions, using proteomics, genomics, live cell imaging, in-cell ELISA and confocal microscopy. The model presented in this article enables other researchers to establish an in vitro model of the bovine alveolus that is easy to set up, malleable and serves as a comparable alternative to in vivo models, whilst allowing study of early host-pathogen interactions, currently not feasible in vivo. The model therefore achieves one of the 3Rs objectives in that it replaces the use of animals in research of bovine respiratory diseases.
摘要：
肺的上皮衬里通常是宿主和吸入病原体之间相互作用的第一个点，过敏原和药物。因此，上皮细胞是旨在阐明宿主-病原体相互作用的研究的主要焦点，解剖局部宿主免疫机制并研究毒理学。如果这些研究不是只在体内进行，开发体外-体内相关性高的体外模型是必要的。我们在这里描述了牛肺泡的共培养模型，旨在克服单一培养和活体动物模型遇到的一些限制。我们的系统包括以24孔Transwell格式接种到可渗透膜上的牛肺动脉内皮细胞（BPAECs）。用永生化的牛肺泡II型上皮细胞覆盖BPAEC，并在使用前在气液界面培养14天；在我们的案例中，研究宿主与分枝杆菌的相互作用。新型细胞系和共培养模型的表征提供了令人信服的证据，表明永生化的牛肺泡II型细胞是原代肺泡II型细胞的真正替代品，它们与BPAEC的共培养提供了生理相关的牛肺泡体外模型。共培养模型可用于研究动态的细胞内和细胞外宿主-病原体相互作用，使用蛋白质组学，基因组学，活细胞成像,细胞内ELISA和共聚焦显微镜。本文提出的模型使其他研究人员能够建立易于建立的牛肺泡体外模型，具有延展性，可作为体内模型的可比替代品，同时允许研究早期宿主-病原体相互作用，目前在体内不可行。因此，该模型实现了3Rs目标之一，因为它取代了动物在牛呼吸道疾病研究中的使用。
