Isolation and Expansion of Spontaneously Immortalized Oral Keratinocytes

Gingival tissues from healthy volunteers were obtained at the NIDCR clinic (Clinical Protocol # 06-D-0144). After surgery, fresh tissues derived from the retro molar area of the oral cavity were incubated overnight in trypsin 0.25% (Sigma Aldrich, CA) at 4°C. Next day, the epithelial compartment was mechanically dissociated from the connective tissue and minced to fine fragments. Keratinocytes were then filtered through a 100 µm cell strainer (BD falcon), pellet at 125 g for 5 min at 4°C, resuspended in keratinocyte serum-free culture medium (Invitrogen, CA), and plated on 60 mm dishes kept in a controlled humidity, temperature, and CO₂ environment [25] . Using this procedure, we observed that most human oral epithelial cells can be expanded for a finite number of passages, achieving a replicative senescent state after approximately 55 days, but few cultures escaped cell senescence, and instead became immortal (G.L. Sanchez, K.L, R.C., J.S.G et al., manuscript in preparation). To date, these cells, termed spontaneously immortalized normal oral keratinocytes, NOK-SI, have been cultured for more than 2 years, retaining epithelial morphology, proliferative capacity, and the expression of typical markers such as cytokeratins and E-cadherin. NOK-SI cell line is routinely cultured in Keratinocyte-SFM medium (Gibco, USA) supplemented with BPE and EGF, penicillin, streptomycin, and maintained at 37°C in a 5% CO2-humidified incubator.