Characterization of a serum-free culture system comparing growth factor requirements of transformed and untransformed cells

We describe the first completely serum-free model culture system for comparing growth control in transformed and untransformed cells. Continuous maintenance of untransformed AKR-2B fibroblasts and chemically transformed AKR-MCA cells in the presence of serum-free medium containing epidermal growth factor (E), insulin (I), and transferrin (T) resulted in cell lines which proliferated with similar doubling times (14 h), comparable to parental lines maintained in 10% serum (16 h). The transformed MCA-SF cells and untransformed AKR-SF cells did not differ in their saturation densities in medium containing E + I + T. However, the monolayer proliferation of MCA-SF cells was significantly greater than that of the AKR-SF cells in the presence of E + T, I + T, or T alone. Both cell lines required T to proliferate in monolayer culture. [³H]-Thymidine incorporation experiments and autoradiographic analysis indicated that quiescent MCA-SF cells could reenter the cell cycle by addition of nutrients alone. The combination of E + I + T produced no additional stimulation of DNA synthesis. In contrast, individual polypeptide growth factors (E, I, IGF-I, PDGF, FGF_{a or b}, or TGF-β₁) were required to elicit a mitogenic response in the untransformed AKR-SF cells. Peak mitogenesis occurred from 18-20 h for all growth factors except TGF-β₁ (32 h). Neither AKR-SF nor MCA-SF cells could grow with anchorage independence in serum-free medium, unless both TGF-β₁ and FGF_{a or b} were simultaneously present. The results indicate that this well-defined, serum-free model system can be utilized to detect growth factor-related alterations associated with the transformed state.