Simian virus 40-transformed 3T3 cells are dependent on serum for survival and growth. This growth activity can be separated on a pH 2 Sephadex G100 column into two fractions: a high molecular weight activity and a low molecular weight substance that has recently been characterized as containing as its major agent, biotin. To replace the remainder of the serum requirement, hormones and other growth factors were tested. Both insulin at high, nonphysiological concentrations (200 to 500 ng/ml) and transferrin (5 X 10(-8) M) stimulate the growth rate in low serum medium (0.3% v/v bovine calf serum DME) individually and, when added together, are nearly as growth enhancing as 10% serum. The need for the residual serum in this medium can be eliminated by the use of crystalline trypsin during trypsinization. Under these serum-free conditions, biotin and transferrin supplementation provide for moderately good growth (20 to 30 hr population doubling time, 1 X 10(6) cells/3.2-cm dish final cell density). Insulin addition further stimulate the growth rate (16 to 20 hr) and the final density (1.5 X 10(6) cells). Although the protein growth factors, EGF (0.5 to 1.0 ng/ml) and FGF (4 to 10 ng/ml), also appear to enhance growth individually and additively, their effects are slight and very variable. Nevertheless, the complete serum-free medium (DME supplemented with biotin, transferrin, insulin, EGF, and FGF) yields growth comparable but still inferior to 10% serum supplementation (14-versus 12-hr population doubling time, 1 to 2 X 10(6) versus 2 to 3 X 10(6) cells final cell density).
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