Colony growth in agar by human melanoma cells

Colony-forming capacity of human melanoma cells was investigated in five established cell lines and isolated cells from seven metastatic or recurrent tumors. A single-cell suspension was added to 0.3% agar medium, and colonies were observed over 7 to 21 days. With the five cell lines, colony formation occurred in all cases and was usually accelerated by 2-mercaptoethanol, but there was wide variation in cloning efficiency between the cell lines (5 to 80%), in the appearance of the colonies, and in their recloning capacity. In those cell lines with low cloning efficiency, some colonies grew attached to the culture dishes, and these had a high recloning capacity. In the melanotic cell lines, colonies originated from slightly or nonpigmented cells which became more pigmented during incubation. Stimulation of colony growth by 2-mercaptoethanol led to reduced pigmentation of colonies, whereas theophylline caused increased melanin production and decreased colony size. With freshly obtained tumor specimens, both the frequency of colonies (0.01 to 0.26%) and the growth rate were much reduced compared to the cell lines. Both could be increased by addition of 2-mercaptoethanol to the medium or by separation of the tumor cell suspension on a density gradient before culture.