0.4 μm pore size polyester membrane

A transwell assay was carried out using the 6-well polystyrene transwell plate with a 0.4-μm-pore-size polyester membrane (Corning, NY). With this permeable membrane, secretions or metabolite, but not bacterial cells in the supernatant of the upper chamber were allowed into the lower chamber where the biofilm formed. 3 ml of TSB broth was inoculated with 30 μl of an overnight culture of ΔtolC in the lower chamber, and 1 ml WT suspension (1/100 dilution) in the upper chamber. Control wells were set with WT or ΔtolC suspension in both top and bottom wells. Transwell plates were incubated at 37°C for 12h. Bacterial suspension in the upper chamber was removed and the biofilms formed in the lower chamber were stained and quantified using crystal violet staining. This experiment was repeated independently three times with triplicate samples.

Transwell® cultures were created with the cervical epithelial and stroma cells using 6.5 mm diameter inserts with 0.4 μm pore size polyester membrane (Corning, Inc.). The surfaces of the porous membrane were coated with collagen IV in the apical side and Collagen I and fibronectin on the basal side following the same method described for the Chip culture. The cell seeding density was adjusted for the TW inserts due to their larger surface area compared to the chip to allow for a comparable cell seeding density in both culture systems. Primary cervical fibroblasts (P5, 0.05 × 10⁶ cells/ml) were first seeded on the basal side of the porous membrane by inverting the insert and 2 h incubation in fibroblast cell growth medium, followed by flipping the Transwells in the well plate and seeding the primary cervical epithelial cells (P5, 0.5  10⁶ cells/ml) on the apical side for 3 h in epithelial cell growth medium. The medium in each chamber of the Transwell was refreshed with respective medium and the inserts were incubated at 37 °C, 5% CO₂ under static conditions. The media of the Transwell inserts were changed every day throughout the culture time following the same media composition change regimen described for the chip culture.

ECs at a density of 50,000 per well were seeded in the upper chambers (0.4 μm pore size polyester membrane from Corning, Inc.) and cultured for 2 to 3 days to produce a confluent monolayer, and MSCs were seeded in the lower chambers,. Then, cells were treated with LPS (100 ng/mL, Sigma) for 6 hours before permeability was tested, as previously described [23 (link)]. After adding 10 μL 40 kDa fluorescein isothiocyanate- (FITC-) Dextran (Sigma-Aldrich) to each upper insert and incubating for 40 minutes in an incubator, 100 μL medium from the upper and lower chambers was withdrawn. Then, the medium was transferred to a 96-well plate and read using excitation and emission wavelengths of 490 nm and 530 nm, respectively.