Dmem ham s f12 3 1

The stored biofilms were thawed on ice, centrifuged and dispersed in Hanks' balanced salt solution (HBSS, Sigma-Aldrich) (Spano et al., 2016 (link)). A sample of biofilm was processed to determine CFU count. The remaining biofilm was applied topically onto RHG, with ~1 × 10⁷ CFU biofilm cells concentrated in a drop of 10 μl in HBSS as previously described (Buskermolen et al., 2017 (link)). The biofilm-RHGs were further cultured at the air liquid interface in medium containing DMEM/Ham's F12 (3/1) (Gibco, Grand Island, USA) supplemented with 1% Fetal Clone III (RHG, Logan, UT, USA), 0.1 μM insulin (Sigma-Aldrich, St. Louis, MO, USA), 2 μM hydrocortisone (Sigma-Aldrich), 1 μM isoproterenol (Sigma-Aldrich), 10 μM carnitine (Sigma-Aldrich), 10 mM L-serine (Sigma-Aldrich), 0.4 mM L-ascorbic acid (Sigma-Aldrich), and 2 ng/ml epidermal growth factor (Sigma-Aldrich), for 24 h at 37°C, 7.5% CO₂ and 95% humidity. After 24-h exposure RHG were harvested.

Immortalized gingiva keratinocyte (KC-TERT, OKG4/bmi1/TERT, Rheinwald laboratory, Boston, USA) (passage 10 to 20) and fibroblast (Fib-TERT, T0026, ABM, Richmond, BC, Canada) (passage 10 to 20) cell lines were used for constructing RHG. The RHG model, consisting of a fibroblast-populated collagen hydrogel (collagen I derived from rat tail) overlaid with keratinocytes (0.5 x 10⁶ cells) was cultured exactly as previously described (Kosten et al., 2015 (link)). In brief, RHG were cultured in six-well transwell inserts (pore size 0.4μm, 24mm, Corning, USA), first submerged in culture medium for 3 days and then lifted to the air-liquid interface for an additional 10 days to induce epithelial differentiation and stratification in culture medium containing DMEM/Ham’s F12 (3/1) (Gibco, Grand Island, USA) supplemented with 1% Fetal Clone III (HyClone, GE Healthcare, Chicago, USA), 1% PS (Gibco, Grand Island, USA), 0.1µM insulin, 1µM hydrocortisone, 1µM isoproterenol, 10µM L-carnitine, 10mM L-serine, 0.4mM ascorbic acid, and 2ng/mL epidermal growth factor. All agents were purchased at Sigma-Aldrich (St. Louis, USA) when not specified. One day prior to bacteria exposure, PS and hydrocortisone were omitted in RHG culture medium for some experimental conditions as indicated below.