The OSSN cells that were used in this study were derived from Caucasian men (age range, 60–82 years) who presented with right limbal lesions, histopathologically diagnosed as either full-thickness dysplasia (severe,
n = 2) or squamous cell carcinoma in situ (
n = 1). Human dysplastic (DCECs;
n = 3) and normal conjunctival epithelial cells (NCECs;
n = 2) were grown from tissue explants and established in pure long-term cultures, according to previously optimized protocols.
12 22 32 In brief, freshly resected diseased and normal conjunctiva was cut into 1- to 2-mm
2 segments, placed in six-well culture plates (Greiner bio-one, Frickenhausen, Germany) and incubated at 37°C in a humidified incubator set to 5% CO
2 in Eagle’s minimum essential medium (EMEM) containing 10% FBS (ThermoTrace, Melbourne, Australia), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM
l-glutamate (ThermoTrace). Explants were removed after 10 days, cells subcultured weekly and purity established after three to four generations with a pan-cytokeratin marker (MNF-116
FITC; Dako Cytomation) and p63 (Clone 4A4; Santa Cruz Biotechnology, Santa Cruz, CA). Mouse IgG
1 FITC (Dako Cytomation) and mouse IgG
2a FITC (Jackson ImmunoResearch, West Grove, PA) were used as appropriate isotype control antibodies for the flow cytometric analysis.
32 Primary cultures reached replicative senescence between passages 15 and 20.