The growth requirements of SV40 transformed Balb/c-3T3 cells have been studied in the absence of serum. For growth in serum-free medium, the cells require (i) insulin, (ii) transferrin, and (iii) cis-unsaturated fatty acids added in combination with fatty acid free bovine serum albumin. The growth rate, saturation density, and morphology of cells grown in this serum-free medium are the same as those of cells grown in serum supplemented medium. This mixture also supports the growth of SV40 transformed Swiss-3T3 cells and SV40 transformed primary mouse embryo cells, but does not support the growth of untransformed Balb/c-3T3 cells. The addition of fibronectin to this mixture allows routine subculture, repeated passage, and indefinite propagation of SV40 transformed Balb/c-3T3 cells. Cells grown in this medium for a period of two months retain their ability to induce tumors when injected into athymic nude mice.

ABSTRACT. Human keratinocytes have been serially cultivated in low (0.015 mM) and high (1.8 mM) calcium containing medium. The calcium concentration of the growth medium significantly influenced the cell growth period in vitro. Cells grown in low calcium medium underwent 35-40 population doublings over 16-17 passages, while cells grown in high calcium medium ceased to proliferate after 20 population doublings over 7 passages. Changing the keratinocytes from one in vitro environment to the other drastically altered the lifespan in culture of populations derived from the same primary tissue. The degree of DNA methylation of human keratinocytes was shown to decrease with age in both high and low calcium culture conditions but does not appear to be associated with differentiation.

ABSTRACT.Calf thymocytes, freshly suspended in a protein-free minimum essential medium quickly lost viability at 3 x 105 but not at 1 x 107 cells/ml. For technical reasons [3H]thymidine incorporation into DNA was routinely used as a relative index of cell viability. The non-dialyzable fraction of a conditioned medium and of a cell-free extract obtained from thymocytes reversed the loss of viability at lower population density. An effective assay system was devised aiming at the isolation of the active factor. Calf serum and simple proteins like serum albumin and cytochrome c were without effect, and the former did not affect the activity of the factor. However, the factor was not highly species-or organ-specific. The abundance of the factor in thymocytes seemed to vary much less than the responsiveness to the factor of thymocytes from different animals.