| Autor: |
Sharon L. Wong, Renee Whan, Nikhil T. Awatade, Adam Jaffe, Alexander Capraro, Shafagh A. Waters, Nihan Turgutoglu, Iveta Slapetova, Elvis Pandzic, Ling Zhong, Laura K. Fawcett |
| Rok vydání: |
2020 |
| Předmět: |
|
| DOI: |
10.1101/2020.05.29.120006 |
| Popis: |
BackgroundPatient-derived airway cells differentiated at Air Liquid Interface (ALI) are valuable models for Cystic fibrosis (CF) precision therapy. Advances in culture techniques have improved expansion capacity of airway basal cells, while retaining functional airway epithelium physiology. However, considerable variation in response to CFTR modulators is observed even when using similar ALI culture techniques. We aimed to address if variation in response reflects true biological differences between patients or technical differences as a consequence of different culture expansion methods.MethodsNasal epithelial brushings from 14 individuals (CF=9; non-CF=5) were collected, then equally divided and expanded under conditional reprogramming culture (CRC) and feeder-serum-free “dual-SMAD inhibition” (SMADi) methods. Expanded cells from each culture were differentiated with proprietary PneumaCult™-ALI media. Morphology (Immunofluorescence), global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility, and ion transport) were compared in CRCALIand SMADiALIunder basal and CFTR corrector treated (VX-809) conditions.ResultsNo significant difference in the structural morphology or global proteomics profile were observed. Barrier integrity and cilia motility were significantly different, despite no difference in cell junction morphology or cilia abundance. Epithelial Sodium Channels and Calcium-activated Chloride Channel activity did not differ but CFTR mediated chloride currents were significantly reduced in SMADiALIcompare to their CRCALIcounterparts.ConclusionAlteration of cellular physiological functionin vitrooccurs were more prominent than structural and differentiation potential in airway ALI. Since culture conditions significantly influence CFTR activity, this could lead to false conclusions if data from different labs are compared against each other without specific reference ranges. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
