Methods to culture human pleural mesothelial (NHM) cells have been defined. Cultures are initiated by pelleting the mesothelial cells from pleural effusion fluid and inoculating the resuspended cells into dishes containing LHC basal nutrient medium supplemented with serum (3%), hydrocortisone (0.5 micromoles), insulin (5 micrograms/ml), epidermal growth factor (EGF) (5 ng/ml), transferrin (10 micrograms/ml), trace elements, and 2% chemically- reduced (factor-free) serum (FFS). Using this protocol, mesothelial cell cultures have been established from more than 200 donors. The cells can be subcultured at clonal density with a colony-forming efficiency of more than 10% and high density cultures can be subcultured four to six times before senescence. We have now established that many factors
