Development Of A Differentiated Procine Tracheal Epithelium Cell Culture Model In Air-liquid Interface

Respiratory epithelial cells are target of the infection of several respiratory pathogens.However,there is a lack of a good model in vitro to study the porcine respiratory pathogens.The airway epithelial cells cultures were grown in an air-liquid interface conditions with media containing defined hormones and growth factors.The cultures will be a pseudo-stratified muco-ciliary epithelium with ciliated cell,secretory cell and tight junction.The purpose of this study is to develop a differentiated porcine tracheal epithelium cell culture model in air-liquid interface and make it standardized by the screening and optimization of culture conditions and methods.Donor pigs are selected considering the health status,ages,strains.The epithelial cells were harvested from tracheal tissue of negative hybrid pigs without specific pathogen to isolate tracheal tissue using pronase and DNase release treatment,through observated the cell adherence ability and the time of establishing ALI.Cell cultures in air-liquid interface condition with different the source of PTEC cells,isolating time,cell density,composition of additives,clture system,they effect cell viability,proliferation and differentiated.We assasy the reliability of PTEC in vitro differentiated model using indirect immunofluorescence assay,epithelial cell electrophysiological characteristics and scanning electron microscope.PTEC are seeded on 0.4μm porous support membrane which coated human placental collagen IV,and the seeding density is 2.67-3.91 ×106cells/cm2.Under these conditions,the cell adherent rate was high and could establish ALI.When creating ALI,cells were polygonal cobblestone;EGF and FBS can help the cells to adhere,but it is inhibition of cell proliferation,thus affecting the time of establishing ALI.The optimal conditions are that growth medium is the BEGM with 15 ng/ml EGF,the differentiation medium is the BEGM added 15 ng/ml and 2%USG.Through analyzing the results of TEER and SEM,the cultures form the cilia and tight junction,and the maximum of TEER is 1838±226Ωcm2.With the optimized conditions,the cryopreserved PTEC was cultured,and there was also a generation of ciliated cell and tight junction.In this study,a differentiated porcine tracheal epithelium cell culture model in air-liquid interface cells and was developed,and further make it standardized.