MAJOR TECHNICAL ADVANCES Pharmacological Rescue of Conditionally Reprogrammed Cystic Fibrosis Bronchial Epithelial Cells Martina Gentzsch 1,2 , Susan E. Boyles 2 , Chaitra Cheluvaraju 2 , Imron G. Chaudhry 2 , Nancy L. Quinney 2 , Crescentia Cho 2 , Hong Dang 2 , Xuefeng Liu 3 , Richard Schlegel 3 , and Scott H. Randell 1,2 1 Department of Cell Biology and Physiology and 2 Marsico Lung Institute/Cystic Fibrosis Research Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and 3 Department of Pathology, Georgetown University School of Medicine, Washington, District of Columbia Abstract Well-differentiated primary human bronchial epithelial (HBE) cell cultures are vital for cystic fibrosis (CF) research, particularly for the development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs. Culturing of epithelial cells with irradiated 3T3 fibroblast feeder cells plus the RhoA kinase inhibitor Y-27632 (Y), termed conditionally reprogrammed cell (CRC) technology, enhances cell growth and lifespan while preserving cell-of-origin functionality. We initially determined the electrophysiological and morphological characteristics of conventional versus CRC-expanded non-CF HBE cells. On the basis of these findings, we then created six CF cell CRC populations, three from sequentially obtained CF lungs and three from F508 del homozygous donors previously obtained and cryopreserved using conventional culture methods. Growth curves were plotted, and cells were subcultured, without irradiated feeders plus Y, into air–liquid interface conditions in nonproprietary and proprietary Ultroser G–containing media and were allowed to differentiate. Ussing chamber studies were performed after treatment of F508 del homozygous CF cells with the CFTR modulator VX-809. Bronchial epithelial cells grew exponentially in feeders plus Y, dramatically surpassing the numbers of conventionally grown cells. Passage 5 and 10 CRC HBE cells formed confluent mucociliary air–liquid interface cultures. There were differences in cell morphology and current magnitude as a function of extended passage, but the effect of VX-809 in increasing CFTR function was significant in CRC-expanded F508 del HBE cells. Thus, CRC technology expands the supply of functional primary CF HBE cells for testing CFTR modulators in Ussing chambers. Keywords: cystic fibrosis; electrophysiology; human bronchial epithelial cells; in vitro models Clinical Relevance Primary cystic fibrosis human airway epithelial cells are vital for development of cystic fibrosis transmembrane conductance regulator modulator drugs, but their supply is limited. These studies demonstrate that coculture with irradiated feeder cells and a Rho kinase inhibitor enables massive expansion of cells useful for testing cystic fibrosis transmembrane conductance regulator modulators in Ussing chambers. Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an anion channel vital for normal transepithelial electrolyte and fluid transport in multiple organs. CFTR is synthesized at the endoplasmic reticulum and is trafficked to the apical epithelial cell membrane where it regulates luminal surface properties. Loss of functional CFTR in the airways results in thick, viscous mucus, impaired mucociliary clearance, chronic infection, inflammation, and tissue damage. The z2,000 known CFTR mutations have variable effects on RNA production and protein synthesis, folding, stability, cellular trafficking, and channel function (1). ( Received in original form August 26, 2016; accepted in final form December 11, 2016 ) This work was supported by Cystic Fibrosis Foundation (CFF) GENTZSCH15XX0, National Institutes of Health P30 DK065988, and CFF BOUCHE15R0. Author Contributions: Conception and design: M.G., C. Cheluvaraju, X.L., R.S., and S.H.R.; performance of experiments: M.G., S.E.B., C. Cheluvaraju, I.G.C., N.L.Q., C. Cho, and S.H.R.; data analysis and interpretation: M.G., S.E.B., C. Cheluvaraju, I.G.C., N.L.Q., C. Cho, H.D., and S.H.R.; drafting the manuscript for important intellectual content: M.G., C. Cheluvaraju, and S.H.R.; and critical review of the manuscript: all authors. Correspondence and requests for reprints should be addressed to Scott H. Randell, Ph.D., The University of North Carolina at Chapel Hill, Marsico Lung Institute/ Cystic Fibrosis Research Center CB 7248, 1117 Marsico Hall, 125 Mason Farm Road, Chapel Hill, NC 27599-7248. E-mail: randell@med.unc.edu This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 56, Iss 5, pp 568–574, May 2017 Copyright © 2017 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2016-0276MA on December 16, 2016 Internet address: www.atsjournals.org 568 American Journal of Respiratory Cell and Molecular Biology Volume 56 Number 5 | May 2017