Porous polyester membrane inserts

HLECs were grown to confluence on porous polyester membrane inserts (12 mm diameter, 0.4 μm pore size; Transwell, Corning, Cambridge, MA). The upper and lower compartments contained 0.5 and 1.5 mL of media, respectively. Monolayers were either left untreated or Poly(I:C) was added to the upper compartment at 0.1, 1 or 10 μg/mL. TEER measurements were performed using an EVOM volt-ohmmeter connected to a 12-mm Endohm unit (World Precision Instruments, Sarasota, FL). A decrease in TEER indicates an increase in monolayer permeability, whereas an increase in TEER signifies an increase in monolayer integrity. Data were collected from triplicate inserts per treatment in each experiment. Values were reported as the percentage of basal TEER obtained by dividing the resistance values of each treated monolayer by the resistance value of the control monolayer at each given treatment.

The Transwell assay was performed as previously described^{12 (link)} on Transwell 12-well plates with porous polyester membrane inserts (0.4 µm pore size; Corning). The membrane inserts were coated with 1% Geltrex diluted in DMEM/F12 for 1 h. For amnion-like cell induction, primed hPSCs were seeded on membrane inserts at a density of 3 × 10⁴ cells per cm² under mTeSR plus 10 μM Y27632. Then, 18 h after cell seeding, the medium was switched to E6 supplemented with bFGF (20 ng ml⁻¹) and BMP4 (50 ng ml⁻¹) and cultured for 48 h. For G6-nHyCs and 7F-nHyCs, day 3 PDGFRA⁺ cells were sorted and recultured on membrane inserts at a density of 9 × 10⁴ cells per cm² overnight. Primed hPSCs were collected as small clumps and seeded onto the membrane inserts under E6 medium supplemented with bFGF (20 ng ml⁻¹). The cells were cultured for another 48 or 96 h before analysis. The medium was changed every other day.