Ability of mammalian fibroblasts to grow in synthetic medium containing neither serum nor exogenously added macromolecules.

Rous sarcoma virus transformed Chinese hamster fibroblasts, clone CHR1-3, were established at high temperature, then subcloned. Six subclones with round and flat morphology harboring undeleted and partially deleted RSV proviruses, respectively, were seeded into serum-free synthetic medium with no macromolecular additives, and maintained for 2 mo. One flat subclone no. 14, fully designated sfCHR1-3.14 for its serum-free phenotype, was further propagated in the same medium. The cells grew exponentially in loosely attached monolayers and could be serially passaged on bare polystyrene with an average population doubling time of 46 h. Cell attachment could be improved by using collagen-coated polystyrene or by adding a methionine supplement to the culture medium. Furthermore, the sfCHR1-3.14 cells could be subcloned and further grown in nonselective medium. The reversion rate of the sf phenotype was estimated to be 1 to 2%/cell generation. Evidence for an autocrinal stimulation was obtained by cloning efficiency assays showing a requirement for a threshold cell density. Slight growth stimulation could also be detected in assays using conditioned medium from sfCHR1-3.14 cells and serum-restricted wild-type (wt)NIH3T3, but not wtCHR1-3.14, cells as indicator cells. Finally, wtNIH3T3 cells used in these assays were assayed for serum-free growth and found to be able to develop their own sf phenotype; in this respect they resemble the previously established sfCHR1-3.14 cells.