Isolation and culture of thymic epithelial cells (TECs) using
conventional primary tissue culture techniques under conditions employing
supplemented low calcium medium yielded an immortalized cell line derived
from the LDA rat (Lewis [Rt1l] cross DA
[Rt1a]) that could be manipulated in vitro. Thymi
were harvested from 4–5-day-old neonates, enzymically digested using
collagenase (1 mg/ml, 37°C, 1 h) and cultured in low calcium
WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10
nM), epidermal growth factor (10 ng/ml), insulin (10 μg/ml),
transferrin (10 μg/ml), 2% calf serum, 2.5% Dulbecco's
Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic.
TECs cultured in low calcium displayed round to spindle-shaped morphology,
distinct intercellular spaces (even at confluence), and dense
reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed
cobblestone-like confluent monolayers that were resistant to
trypsinization (0.05%) and displayed keratin intermediate filaments
concentrated at desmosomal junctions between contiguous cells. Changes in
cultured TEC morphology were quantified by an analysis of
desmosome/membrane relationships in high and low calcium media.
Desmosomes were significantly increased in the high calcium medium. These
studies may have value when considering the growth conditions of cultured
primary cell lines like TECs.