Population-dependent requirements of vitamin B12 and metabolically related substances of several mouse cell types in serum-free, albumin-fortified medium.

Mouse FM3A cells propagated well in serum-free medium containing 0.5% serum albumin and 1 microgram of insulin/ml. The vitamin B12 (B12) requirement of the cells depended on the population density. This requirement disappeared when a sufficiently large cell population was present. A combination of 1-100 ng of B12/ml and 4 micrograms of hypoxanthine/ml resulted in a synergistic increase in cell growth at low cell densities. A similar growth response was obtained when the B12 plus hypoxanthine was replaced by 4 micrograms of hypoxanthine/ml in combination with 100 ng of thymidine/ml, 1 microgram of folic acid/ml or 1 microgram of folinic acid/ml, even though 1 microgram of folic acid/ml already was present in the medium. Experiments on single cell inoculation showed that colony size and the yield of cells grown in B12-supplemented medium were much larger than those for cells grown in B12-free medium. A more critical population-dependent B12 requirement was demonstrated in mouse Ehrlich and L cells and their hybrids. At less than 100 cells there was no propagation in serum-free medium lacking B12, folinic acid and thymidine; whereas, a satisfactory growth response was obtained in medium supplemented with these substances.