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Abstract
Primary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. pBECs collected from healthy donors (n = 5) were CR using g-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCult (PN-ALI) or bronchial epithelial growth medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, Lonza)—(AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral RNA was assessed by RT-qPCR and anti-viral proteins quantified by LEGENDplex following Rhinovirus-A1b infection. CRpBECs differentiated in PneumaCult were smaller and had a lower TEER and cilia beat frequency compared to BEGM media. PneumaCult media cultures exhibited increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. There are distinct structural and functional differences in pBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing CRpBECs ALI experiments for specific research questions.
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1 University of Newcastle, School of Medicine and Public Health, Callaghan, Australia (GRID:grid.266842.c) (ISNI:0000 0000 8831 109X); University of Newcastle, Immune Health Program, Hunter Medical Research Institute, New Lambton Heights, Australia (GRID:grid.266842.c) (ISNI:0000 0000 8831 109X)
2 University of Newcastle, School of Medicine and Public Health, Callaghan, Australia (GRID:grid.266842.c) (ISNI:0000 0000 8831 109X); Hunter Medical Research Institute University of Newcastle, Asthma and Breathing Research Program, New Lambton Heights, Australia (GRID:grid.413648.c)
3 University of Newcastle, School of Medicine and Public Health, Callaghan, Australia (GRID:grid.266842.c) (ISNI:0000 0000 8831 109X); Hunter Medical Research Institute University of Newcastle, Asthma and Breathing Research Program, New Lambton Heights, Australia (GRID:grid.413648.c); John Hunter Hospital, Dept of Respiratory and Sleep Medicine, New Lambton Heights, Australia (GRID:grid.414724.0) (ISNI:0000 0004 0577 6676)
4 University of Newcastle, Immune Health Program, Hunter Medical Research Institute, New Lambton Heights, Australia (GRID:grid.266842.c) (ISNI:0000 0000 8831 109X); Centenary Institute and University of Technology Sydney, Centre for Inflammation, Sydney, Australia (GRID:grid.117476.2) (ISNI:0000 0004 1936 7611)
5 University of Newcastle, School of Medicine and Public Health, Callaghan, Australia (GRID:grid.266842.c) (ISNI:0000 0000 8831 109X); University of Newcastle, Immune Health Program, Hunter Medical Research Institute, New Lambton Heights, Australia (GRID:grid.266842.c) (ISNI:0000 0000 8831 109X); Hunter Medical Research Institute University of Newcastle, Asthma and Breathing Research Program, New Lambton Heights, Australia (GRID:grid.413648.c); John Hunter Hospital, Dept of Respiratory and Sleep Medicine, New Lambton Heights, Australia (GRID:grid.414724.0) (ISNI:0000 0004 0577 6676)