Human embryonic diploid lung fibroblasts were capable of growing in a medium consisting of Eagle's MEM vitamins plus 0.1 µg/ml biotin, Earle's salt solution, 4 mM glutamine, 0.4 mM cystine, 0.5 mM tyrosine, 10 % undialysed calf serum and 1 mM pyruvate. The growth rate and percent cell attachment in this medium was slightly less than in Eagle's MEM but the longevity was about the same. Addition of 0.5-1.0 mM histidine stimulated growth but addition of O.2 mM phenylalanine, methionine or valine decreased longevity markedly.
Maintaining a constant pH of about 7.5-7.6 with 20 mM of the organic buffers HEPES, TES or TRICINE yielded a growth rate equal to or better than that of cells growing in unbuffered medium. Some buffers gave good growth initially but eventually the longevity decreased (20mM BIS-TRIS, 20 mM BICINE, 10 mM TRIS). In almost all cases the initial exposure to the buffers yielded the best growth results.