Methods to culture human pleural mesothelial (NHM) cells have been improved. Pure 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 pseudo-defined protocols, 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 transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF) will induce serum-starved cells to undergo one round of DNA synthesis in the absence of serum. However, for sustained growth to ensue, the medium must also be supplemented with insulin and high density lipids (HDL). We have further found FFS to be both a good source of HDL and essentially free of other growth factor activities. Surprisingly, we have found that NHM cultures, on average, respond equally well in mitogen assays to numerous purified peptide growth factors including: interleukin 1, interleukin 2, EGF, fibroblast growth factor, PDGF, TGF-beta, beta-interferon, gamma-interferon and cholera toxin. Further, insulin is required for sustained growth and transferrin potentiates the activities of the other factors.
