"Contact inhibition" of cell division in 3T3 cells.

The 3T3 cell, an established line of mouse fibroblast cell, has been considered to be extremely sensitive to "contact inhibition" of cell division. Under the usual culture conditions, 3T3 cells grow rapidly in sparse culture, but cell division stops after the cells become confluent, at approximately 106 cells per 6-cm dish. The cell monolayer has a typical "cobblestone" appearance. Todaro, Lazar, and Green1 have described studies of the effect of serum on cell division in these "contact-inhibited" cells. The addition of serum to an inhibited culture leads to a rise in RNA synthesis, followed in a few hours by protein synthesis, and eventually by some DNA synthesis. Todaro, Lazar, and Green1 have inferred that a factor in serum overcomes "contact inhibition" of cell division. In the experiments described below, we find that the characteristic "contact-inhibited" cell density observed for 3T3 cells is a fortuitous result of growing the cells in a medium that contains 10 per cent calf serum. The final cell density, after cell division stops, is directly proportional to the amount of serum added to the medium.2 The experiments suggest that serum contributes a factor or factors required by 3T3 cells for cell division. Viral-transformed 3T3 cells have a greatly reduced requirement for the serum factor(s). Whether serum factors offer an explanation of "contact inhibition" of cell division in other instances remains to be determined. It is pertinent that Temin3 has found that an insulin-like factor in serum is required for cell divison by cultured chick cells. Transformation of chick cells by Rous sarcoma virus lowers the requirement for this serum factor. Materials and Methods.-The 3T3 cell line was obtained from Dr. Marguerite Vogt. The cell line had been obtained originally from Dr. Howard Green and had been cloned recently by Dr. Vogt to maintain the typical "cobblestone" appearance. During prolonged culture, 3T3 cells gradually lose their high requirement for serum and grow to higher cell densities. Therefore, the cell line was maintained at 90'C, and cells used in the experiments were not cultured over 8 weeks. The cells were grown in enriched Eagle's medium, as used in Dulbecco's laboratory.4 To count the cells, the medium was removed from the dishes, the cell layer was trypsinized with half the concentration of trypsin used during transfer of the cells, and the cells were counted in the trypsin solution by means of a hemacytometer. Counts were on duplicates. Experiments were replicated at least three times. The standard error observed for replicate counts was approximately 10%. Assay for growth factor: Approximately 105 3T3 cells were plated per 6-cm plastic dish in 5 ml of medium with 6% calf serum. Solutions to be assayed were added 24-48 hr later. (The solutions to be assayed were sterilized by 3-min UV irradiation with a germicidal lamp rather than by filtration, since the growth factor appears to be adsorbed on Millipore filters under some conditions.) Counts of the final number of cells per dish were made at 5 days, usually about a day after growth had stopped. Partial purification of the growth factor from human urine: The urine was frozen immediately, to avoid microbial action, and was lyophilized. The residue was dissolved in water to give one tenth the original volume, and the solution, with suspended solids, was dialyzed overnight at 40C against 0.05 N sodium chloride, in the presence of chloroform.