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At least seven compounds synthesized by cultured cells in amounts which should suffice for sustained growth have nevertheless proved under certain conditions necessary for survival (asparagine, cystine, glutamine, homocystine inositol, pyruvate, serine). In every instance so far examined, that requirement has been population-dependent, disappearing at cell densities sufficiently large to bring the concentration in the medium and in the cellular pool to metabolically effective levels before the cells died of the specific deficiency. At population densities of less than 100 cells per ml, serine was required by all cultured cells so far studied. With more exigent strains, such as the RT6 strain of rabbit fibroblast and the P388 mouse leukemia, the serine requirement disappeared only at populations in excess of 50,000 and 150,000 cells per ml, respectively. The requirement for pyruvate by the latter cell as an alternative to serine also disappeared at that population density. In a cystine-free medium there were population-dependent requirements for cystine, homocystine, or serine, depending on the experimental conditions. With methionine and glucose as cystine precursors, the critical population density permitting cellular survival and growth was in excess of 200,000 cells/ml. The provision of homocystine as an intermediate reduced the critical population density to 10,000 to 60,000 cells/ml; with the further provision of serine, the critical cell concentration permitting growth was reduced to 50 to 500 cells/ml. Cells adapted to glutamic acid, and capable of utilizing it as a substitute for glutamine, nevertheless required exogeneous glutamine at cellular densities of less than 50,000 cells per ml. In some experiments, the provision of asparagine reduced the critical population density to 10,000 cells/ml, presumably because of its glutamine-sparing action. Inositol is required by most cell lines, despite their demonstrated capacity to synthesize it from glucose. With at least one cell line (HeLa), sustained growth was occasionally achieved in an inositol-free medium if the population density was maintained in excess of 240,000 cells/ml. The possible implications of these findings with respect to the loss of specific organ functions in dispersed cell culture are discussed.

THE POPULATION-DEPENDENT REQUIREMENT BY CULTURED MAMMALIAN CELLS FOR METABOLITES WHICH THEY CAN SYNTHESIZE