Fib tert

Immortalized human gingiva cell lines (keratinocytes and fibroblasts) were cultured and used to construct RHG exactly as described previously (Buskermolen et al., 2016 (link)): keratinocyte (KC-TERT, OKG4/bmi1/TERT, Rheinwald laboratory, Boston, MA, USA) (Dickson et al., 2000 (link)) and fibroblast (Fib-TERT, T0026, ABM, Richmond, BC, Canada). In short, a collagen solution containing fibroblasts was pipetted into a six-well transwell insert with 0.4 μm pores (Corning). After 3 days culture, keratinocytes (5 × 10⁵ cells/transwell) were pipetted on top of the fibroblast-populated collagen hydrogel. After a further 3 days submerged culture, RHG was lifted at the air—liquid interface and further cultured for 10 days in which time a differentiated, stratified epithelium developed on the fibroblast-populated collagen hydrogel.

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.