Endohm

iBMECs were seeded at a density of 1 × 10⁶ cm⁻² onto Transwells that were treated overnight with 50 μg mL⁻¹ human placental collagen IV (Sigma) and 25 μg mL⁻¹ fibronectin from human plasma (Sigma). Transendothelial electrical resistance (TEER) values (Ω cm²) were recorded using an EndOhm (World Precision Instruments), as previously reported [23 (link)]. All measurements were performed on 6.5 mm Transwells with a 0.4 μm pore polyester membrane insert (Corning). TEER values for Transwells with no cells were subtracted from the measured values, and were then normalized to the membrane area. The effects of antibodies, cancer-conditioned medium, and co-cultured cancer cells on TEER were tested (Additional file 1: Fig. S5). The apical chambers of Transwells were exposed to identical concentrations of the fluorescently-labeled molecules as used in 3D assays for 24 h. Conditioned medium was collected from microvessels as the downstream perfusate and stored at −80 °C. The direct effects of co-cultured cancer cells on TEER were tested by seeding the basolateral chamber of Transwells with ~ 50 cancer spheroids, matching the ratio of iBMECs to spheroids used in 3D models.

Transendothelial electrical resistance (TEER) across the monolayer was measured using an endothelial microvolt-ohmmeter and concentric-ring electrode system (EndOhm, World Precision Instruments; Sarasota, FL). Measurements were performed daily to assess readiness for experimentation, as well as following periods of IPC, OGD, and RGR. After the cell support insert was placed in the ohmmeter, readings were allowed to stabilize for 10 sec before recording a final value. The resistance of a cell-free insert was subtracted from each measurement, and the resulting value was multiplied by the surface area of the membrane support to obtain TEER in Ω × cm². The final TEER value prior to experimentation was considered the “baseline” value, and results are reported as a percentage of baseline.

The 16HBE or A549 cells were seeded in the upper chamber of a Transwell tissue culture plate (12 mm diameter, 0.4 μm pore size, Costar, Corning Inc., Corning, NY, United States) and allowed to reach confluence. The basolateral and apical sides of the filters were exposed to CCL3 (10 mg/ml) when indicated. The TER of the cells grown on filters was measured after 7 days, with an epithelial volt-ohm meter (Endohm; World Precision Instruments, Sarasota, FL, United States). To explore the rapid effect of CCL3 on the TER, the volt-ohm meter was coupled to an A/N converter (World Precision Instruments, Sarasota, FL, United States), and the TER measurement was monitored using Powerlab software (Chart for Windows, v4.0, AD Instruments, Sydney, Australia) with an acquisition frequency of 2 Hz. The background electrical resistance attributed to fluid and a blank Transwell filter were subtracted from the measured TER. The TER measurements were normalized by the area of the monolayer and given as cm². Untreated 16HBE cells have been reported to have a TER around 600 Ω. cm² (Yuan et al., 2020 (link)), while A549 cells have been reported to have a TER around 175 Ω. cm² (Albano et al., 2020 (link)).

Cells were grown to confluence in culture medium on collagen IV-coated transwell permeable supports (0.4 μm polyester membrane, 12 mm insert; Costar, NY, USA), culture medium was replaced with EGM-2 MV (minus the VEGF supplement; Lonza, Basel, Switzerland) for 72 h. TEER was measured before and after inhibitor treatment using a Microvolt-ohmmeter and concentric-ring electrode system (EndOhm, World Precision Instruments; Sarasota, FL, USA). After the transwell was placed in the chamber, readings were allowed to stabilize for 10 s before recording. The value was multiplied by the surface area of the membrane support to obtain TEER in Ω/cm². Once approximately 20–25 Ω/cm² was reached, Stattic/AG490 were added for 12 h. This reading is in accordance with other studies for brain endothelial cells cultured in the absence of astrocytes/pericytes [51 (link)]. Since the resistance of each transwell was not identical before treatment, results were calculated by expressing the post-inhibitor resistance as a percentage of its pre-inhibitor value, normalized to vehicle.

Transepithelial electrical resistance (TEER) measurements were performed using EndOhm (World Precision Instruments, Sarasota, FL, USA). Prior to the measurement, the skin equivalent was incubated inside a biosafety cabinet for 15 min to allow equilibration to ambient temperature and humidity. After washing with D-PBS (−), the skin equivalent was inserted into the EndOhm instrument. Values were recorded beginning at 10 s.