Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

Abstract

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

Choice of Differentiation Media Significantly Impacts Cell Lineage and Response to CFTR Modulators in Fully Differentiated Primary Cultures of Cystic Fibrosis Human Airway Epithelial Cells

“…Even though these in vitro studies employed different growth conditions, whether this explains the discrepant results is not entirely clear. For example, Schultz et al used the UNC growth media, i.e., the same in vitro growth conditions as the current study, and their ASL pH results agree with our previously published results, which again used UNC media, and which also did not show significant differences in the steady-state pH of the ASL at rest between non-CF and CF cultures although the ability of cAMP agonists to increase ASL pH was completely absent in CF cultures [ 53 ]. In addition to CFTR, the activity of the proton ATPase, ATP12A has also been shown to play an important role in ASL pH homeostasis, both in vitro [ 76 ] and in vivo [ 56 ].…”

“…The next day, fluorescence was recorded using a temperature and CO 2 -controlled plate reader (TECAN SPARK 10M, Tecan UK, Theale, UK) and agonists (Forskolin and P5) were added to the basolateral compartment at the indicated times (see figures). The data analysis was performed as previously described [ 53 ]: after subtracting background values from pHrodo and Alexa Fluor ® 488, ratios were generated for each time point and pH was calculated from a standard curve where pH was clamped using highly buffered solutions between 5.5 and 8. To reduce inter-experiment variability, the standard curve calibration was performed at the end of each independent experiment.…”

Transport properties in CFTR−/− knockout piglets suggest normal airway surface liquid pH and enhanced amiloride-sensitive Na+ absorption

“…However, regarding the CF lung disease, intrabronchial pH values are probably more relevant to the disease. Saint-Criq et al performed longterm ASL pH measurements on fully differentiated primary human airway epithelial cells under very stable conditions [44]. ASL pH was found not to be different between non-CF and CF under resting (non-stimulated) conditions, but was enhanced in non-CF after stimulation with forskolin.…”

Measuring cystic fibrosis drug responses in organoids derived from 2D differentiated nasal epithelia

“…As an alternative of determining drug efficacy directly in individuals with CF, the effects of CFTR modulators can be predicted using patient-derived epithelial cultures in functional CFTR assays 5 . This is traditionally done with 2D air-liquid interface (ALI)-differentiated airway epithelia by assessment of CFTR-dependent chloride (Cl -) currents via electrophysiology [6][7][8] or via indirect measurements of CFTR, such as quantification of ciliary function, airway surface liquid depth, or pH [9][10][11] . However, a major disadvantage of the ALI-culture model system is the limited scalability.…”