The nature of conditioning nutrients for human malignant melanoma cultures

From a human melanoma line (MM96), showing some dependence of its rate of growth and cell attachment on serum concentration, sublines were selected for even greater dependence on serum factors. These sublines were used to identify the production of substances by other melanoma cells in culture that would supplement or replace the requirement for serum. Most of the sublines showed higher colony-forming efficiency in medium conditioned by one of several cell types in the presence of a low concentration of serum (2.5%) compared with fresh medium containing a high concentration of serum (10%). The conditioning factor(s) were found to be moderately heat-stable, nonlipophilic, and to be of low molecular weight (less than or greater than 400). Screening of a variety of non-essential low molecular weight nutrients, which have been reported to potentiate the growth of a variety of cell types in low-density culture, was positive for the MM96 sublines only for pyruvate. In particular, L-alanine, L-serine, putrescine and alpha MSH (melanocyte-stimulating hormone) were ineffective. Despite the problems of comparing conditioned media with fresh medium, a reasonable correlation between the stimulatory effect and the cell content of added 2-oxocarboxylates was apparent. As would be anticipated, MM96 cultures showed a population density-dependent enhancement of growth up to a cell density of 2 to 4 x 10(4) cells cm-2. Further increase in the initial cell density of these cultures led to a decline in growth rate. An important additional observation was that simple dilution of the ingredients of RPMI1640 with phosphate-buffered saline or Hanks' balanced salt solution led to a reversal of growth inhibition accompanying a serum shift-down.