Mouse keratinocyte cell lines were obtained without initial crisis by use of a serum-free medium, LEP/MK2 (low-calcium Minimal Essential Medium with non-essential aminoacids, supplemented with eight factors). The lines are subtetraploid with random gain and loss of otherwise normal chromosomes. At higher passage numbers, the lines acquire greater independence from growth factors and become less sensitive to growth inhibitors; the requirement for bovine pituitary extract becomes replaceable with bovine serum albumin or with somatomedin C. Transforming growth factor beta (TGF-beta) is a major growth inhibitor of these lines, without inducing terminal squamous differentiation. Cells exposed to 1000 pg/ml of TGF-beta in medium LEP/MK2, and so maintained, developed into a TGF-beta-resistant cell line (MK/2067C), which remains responsive to induction of terminal squamous differentiation by calcium and by serum. The TGF-beta-resistant cell line is subtetraploid with three marker chromosomes. The effect of carcinogens and/or oncogenes is studied in these cell lines.
