Evom2

Transepithelial electrical resistance (TEER) was measured using an epithelial volt-ohmmeter (EVOM2, World Precision Instruments, Sarasota, FL), following the methods previously described (Chang et al., 2019 (link)). Briefly, the volt-ohmmeter was calibrated using a test electrode prior to the measurements. At time zero and 48 h after treatment with BAP or vehicle, DPBS was added to the apical chamber and the probe was added to both apical and basal chambers and resistance was measured (ohms) for each insert. In order to correct for the background resistance, an empty culture insert with DPBS in the apical and basal chambers was also measured. Four cultures were used for each treatment concentration and time point. Percent TEER of control was calculated by subtracting background from all values, then calculating the difference between each treatment group compared to DPBS vehicle control in normal phenotype cells.

The on-chip TEER measurements were performed using an EVOM2 (World Precision Instruments, Sarasota, FL, USA) at room temperature. The chip was connected to the device via a customized connector with spring pins connected to the gold pads of the microfluidic system. As an off-chip reference, a commercial EndOhm 12 G chamber (World Precision Instruments) was used.

The TEER values were measured using an STX2 chopstick electrode with EVOM2™ (World Precision Instruments, Sarasota, FL, USA), following the manufacturer’s instructions, to evaluate cell layer integrity. The measurements obtained were corrected by subtracting the empty insert value and multiplying it by the surface area of the culture insert (0.33 cm²) to derive the final TEER values.

WT mCCD_cl1 cells and clonal Adam10 KO mCCD_cl1 lines were polarized by growing cells on Corning Costar Snapwell Permeable Support inserts (12 mm, 0.4 μm pore size). Cells were seeded at a 1:1 split ratio and grown for 10 days. On day 8, the cells were fed with basal medium containing charcoal-stripped FBS and Pen–Strep supplements only and on day 9 with basal media containing Pen–Strep only. Measurements for transepithelial voltage (V_te) and transepithelial resistance (R_te) were made with a transepithelial volt-ohm-meter and a set of chopstick “STX” electrodes (EVOM2; World Precision Instruments, Sarasota, FL), and the equivalent short-circuit current (I_sc) was calculated using Ohm's law. By convention, a negative I_sc reflects either electrogenic secretion of cations, electrogenic absorption of anions, or a combination of both. Aldosterone and amiloride (Sigma Life Science) were used at 3 nm and 10 μm, respectively.

Transepithelial resistance (TEER) was measured on days 1 and 3 with an epithelial voltmeter (EVOM2, World Precision Instruments). Media was replaced 30 minutes prior to measurements. Readings were done in triplicate and averaged for each well. Alkaline phosphatase activity was measured with a SensoLyte FDP Alkaline Phosphatase Assay Kit from individual transwells according to manufacturers instructions. Apical to basolateral flux through epithelial monolayers was determined by addition of FITC-coordinated dextran (Sigma) to cell culture media (2.2 mg/ml, Sigma) in the apical transwell chamber. Basolateral media was sampled every 30 min for 2 hours to determine dextran flux. FITC-dextran was quantified on a fluorescent plate reader. Analyses were done in triplicate for 3 separate experiments (alkaline phosphatase and dextran flux) or 6 experiments (TEER).

As described above, the VeraVec (P. 2) cells were seeded on the basal side. After cell attachment, the chip was closed and filled with EGM™-MV2. VeraVec cells were grown on the chip for 2 days (with no medium on the apical side) in EGM™-2MV. Then, the EGM™-2MV medium was exchanged with a 1:1 mixture of EGM™-2MV and SAGM™. After medium exchange, the hAEpCs were seeded on the apical side, as described above (day 0). Cell culture medium was exchanged every second day. TEER measurements started at day 1. TEER was measured daily using a commercially available 96-well plate electrode (STX100M; World Precision Instruments) and an EVOM2. The TEER was measured in submerged cell culture conditions for up to 22 days. The specific design of the lung-on-chip enables to hold the electrodes tightly between the outlet well and the culture well. This allows an accurate and reproducible positioning of the TEER electrodes. To measure TEER, electrodes were placed into the cell culture well and outlet well, and then the valves were opened. TEER background was measured on a porous membrane chip containing no cells. Background-subtracted TEER values (Ω) were multiplied by the surface area (0.07 cm²) to calculate Ω × cm².