Esophageal epithelial cells were recovered from autopsy specimens within 6 hours of death. About 1% of the cells formed colonies upon plating. On subcultivation, the plating efficiency of the cells increased about 8- to 10-fold. Immunohistochemical analysis of cultured esophageal keratinocytes revealed expression of cytokeratins characteristic of basal keratinocytes and of hyperproliferating epithelium.
Segments of a normal human esophagus were harvested within 5–6 hours postmortem. The esophageal mucosa from each sample was stripped from the muscularis propria and incubated in Dispase (2.5mg/ml) for 2 h in order to separate the epithelium from the lamina propria. The epithelium was minced and incubated in a mixture of trypsin-EDTA (0.05%-0.02%) for 30 min. The supernatant was collected and centrifuged at 1,300 rpm for 10 min. The recovered cells were plated onto feeder layers of sublethally irradiated 3T3-J2 mouse fibroblasts in 35 mm Petri dishes at a density of about 3x105 cells/mm2. On primary culture and during all subsequent passages, care was taken not to hold the disaggregated cells in suspension for more than 30 min, in order to avoid initiation of terminal differentiation of normal keratinocytes due to the loss of stratum contact. Isolated cells were grown in a 3:1 mixture of DMEM:Ham’s F12 medium supplemented with FBS (10%), penicillin (60 units/ml), streptomycin (0.04 mg/ml), hydrocortisone (0.2 mg/ml), cholera toxin (10-10M), transferrin (5 mg/ml), triiodo-l-thyronine (2x 10-6 M), insulin (5 mg/ml) and EGF (10 ng/ml). The medium was changed every 3–4 days. After 10 days, multiple small colonies of 6–10 cells/clone were present. At this time, the remaining 3T3 cells were removed using EDTA (0.02%), and the growing keratinocytes were released to single-cell suspensions, using trypsin-EDTA (0.05%-0.02%) solution. Cells were subcultivated under the same conditions. The process was repeated to produce quaternary cultures.