Culture Of Airway Epithelial Cells At An Air-liquid Surface

Human airway epithelial (hAE) cell cultures are instrumental for studying basic and applied aspects of respiratory tract biology, disease, and therapy. When primary epithelial cells from the human nasal passages or tracheo-bronchial airways are grown on collagen coated 0.4um polyester membranes at an air-liquid interface (ALI) they undergo mucociliary differentiation, reproducing both the in vivo morphology and key physiologic processes such as mucous secretion, and cilia beating. The cells could maintain differentiated morphology and physiological function close to in vivo epithelium at an air-liquid interface culture.Now three approaches, animal models、chicken embryo culture and two-dimensional (2D) cell culture, can be performed to isolate virus in vitro. But animal models are costly and are subject to extensive criticism within our society and the use of our distant cousins is limited. Chicken embryo culture and 2D cell culture have advantages compared with other measures. Only a minority of viruses can be isolated by using chicken embryo culture, the application is too limited to carry out the research of the biological characteristics and pathogenesis of the virus. In 2D cell culture system, cells are grown on two-dimensional hard plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the complex three-dimensional (3D) architecture of a tissue or organ composed of various cell types, extracellular matrix and interstitial fluids. As a consequence, many kinds of viruses hardly be isolate by these unnaturally shaped cells, resulting in the poor understanding of pathogenic mechanisms of the viruses. Fortunately, an ALI culture can recapitulate the characteristic pseudostratified mucociliary morphology and key physiologic functions and is a quantum leap towards the in vivo biology. These cultures serve as a critical milestone test of biological relevance. However, ALI culture involves the complex interplay of several partners, cells, extracellular matrices and interstitial fluids, so this method will require more efforts to establish standardisation to ensure reproducibility.In the last thirty years, many infectious diseases have been constantly emerging, causing great damage to people lives and property locally or around in the world. Now we know that more than half of these terrible diseases are caused by viruses such as influenza virus、human Bocavirus (HBoV)、human rhinovirus C (HRV-C)、human coronavirus and human Metaenumovirus (hMPV), part of which are hard to propagate in vitro such as HBoV、HRV-C、HKU1. In order to propagate these viruses in vitro and provide an effective approach to study the host-virus mechanism, we have established a comprehensive approach for ALI culture in our study. Our culture produced a pseudo-stratified epithelium with columnar cells supported on a collagen-treated membrane and a detailed protocol for ALI cultures was summarized. Many kinds of viruses replication in vitro was achieved including HBoV, so our study provided an effective method and technology supports to isolate emerging and reemerging "hard-culture" viruses in vitro.The main results of this study are as follows:1、A comprehensive protocol for ALI culture was established, reproducing the characteristic pseudostratified mucociliary morphology and key physiologic functions in vitro:(1) In our study, highly purified and viable primary airway epithelial cells could be harvested and subcultured by our methods, including human being、mouse and swine. Morphology and cytokeratin immunocytochemistry staining confirmed the nature of the airway epithelial cells.(2) In our study, differentiation of airway epithelial cells from different species was achieved at the air-liquid interface including human being、mouse and swine. Morphology, expression of β-tubulinIV、ZO-1 and Mucin5AC, the development of tight-junctions and epithelium were similar with human well-differentiated, multilayered, columnar epithelium containing both ciliated and goblet cells.2、Many kinds of respiratory virus replication in vitro was achieved in fully differentiated human ciliated airway epithelium. So it laid a foundation for further study of the pathogenic mechanism:(1) Respiratory virus replication in ALI culture:After inoculated with positive sample including influenza virus、adenovirus、HBoV and hMPV, real-time PCRs were performed. Results suggested that all kinds of viruses propagated in our ALI culture system.(2) Virus replication has induced cellular immune response. To further characterize the interaction of HBoV with ALI cuture, we determined the concentrations of a range of cytokines/chemokines in the basolateral medium at 24 and 120 hpi compared with those in mock-infected controls. Of the analytes tested, IL-6、IL-8、IP-10、TNF-a and CCL5 were elevated at 24 hpi. By 120 hpi IL-6、IP-10 and CCL5 elevated significantly.Taking together, the primary cells isolated from different species airways could maintain differentiated morphology and physiological function close to in vivo epithelium at an air-liquid interface culture and thus these cultures are useful for studying respiratory tract biology and airway diseases in our study. Our findings in virus replication and cytokines/chemokines changes may lead to a better understanding of pathogenic mechanisms of respiratory tract diseases.