Establishment And Application Of In Vitro Differentiation Model Of Porcine Airway Epithelial Cells

Background:The differentiation model of human airway epithelial cells has been widely used to identify emerging respiratory viruses,study of host/respiratory pathogen interaction and respiratory system disease.The recognition of the pig’s natural susceptibility to respiratory virus infection and similarities to humans in lung physiology, airway morphology, cell types and cell receptor distribution present in the respiratory tract makes it an ideal animal model for studying the infection of respiratory viruses and drug screening of respiratory system disease.Objective:The aim is to establish in vitro differentiation model of porcine airway epithelial cells, identify the morphological and physiological characteristics of PAEC and investigate the application of exogenous gene transduction.Methods:In the present study, porcine airway epithelial cells were collected after protease XIV digestion at low temperature. Primary cells can be expanded in serum free medium containing a variety of cell growth factors on collagen Ⅰ/Ⅲ-coated plastic dishes. PAEC cells were differentiated on collagen IV-coated semipermeable membrane porous supports. We established this in vitro differentiation model of PAEC by the method of air-liquid interface culture. The cell types, morphological characteristics and the typical cell marker proteins on the surface of the PAEC model were detected by scanning electron micrograph,electrophysiology and immunohistology methods.Recombinant adeno-associated virus holds considerable promise for gene therapy due to the low immunogenicity and the relatively stable expression of transgenes.We transfected three plasmids, including pXX680, pXR6 and PSC-GFP to 293T cells according to the proportion of 6:5:3. Then we harvested 293T cells containing rAAV6-GFP after cultured for 48h. rAAV6-GFP were purified by two rounds of CsCl ultracentrifugation. We infected the well-differentiated PAEC with rAAV6-GFP to investigate the transduction efficiency of AAV6 and verify if AAV6 can be effectively mediated exogenous gene expression.Results:PAEC cultures consist of a multilayer epithelium structure including ciliated cells and mucous secretory cells with basal cells located directly beneath the layer. The electrophysiological properties-trans epithellal electric resistance of well-differentiated PAEC model remain stable in a certain period of time, indicating the formation of connections between PAEC cells and the balance of ion transport across cells. We successfully generated rAAV6-GFP by three plasmids transfection method. rAAV6-GFP can infect PAEC from the apical surface and can efficiently transduce PAEC.Conclusion:The establishment of in vitro well-differentiated model of porcine airway epithelial cells lays a good foundation for the study of respiratory pathogens infection, the drug screening and gene therapy of respiratory system disease.