Isolation and Culture of Gingival Epithelial Cells

Gingival epithelial cells (GECs) were isolated and grown as previously described (4 (link)). Briefly, gingival tissue was obtained from a healthy human adult undergoing third molar extraction. Following washing in calcium- and magnesium-free phosphate-buffered saline (PBS) containing streptomycin-penicillin and Fungizone, 0.3 cm² pieces of tissue were incubated overnight in 0.4% dispase at 4°C. Epithelial strips were then separated by mechanical separation and trypsinized for 10-15 min at 37°C in 0.05% trypsin/0.53 mM EDTA. After vigorous pipetting, the cell suspension was centrifuged for 5 min at 700 rpm and the cell pellet subsequently collected and resuspended in keratinocyte growth medium (Dermalife Basal Medium, Lifeline, Walkersville, MD). KGM is a serum-free medium supplemented with 10 ng/ml epidermal growth factor (EGF), 5 μg/ml insulin, 0.5 μg/ml hydrocortisone, 0.15 mM calcium and gentamicin/amphotericin B. Cells were cultured at 37°C, 5% CO₂, with humidity, and utilized for assays between passages 4-6.