A Protein Growth Factor for Mammalian Cells in Culture

Abstract

The amino acid and vitamin requirements of mammalian cells in culture have been carefully defined in the studies of Eagle and coworkers (1) who supplemented their medium with 5 per cent dialyzed serum.Knowledge concerning the growth requirements satisfied by the serum is completely lacking.In a preliminary report (2), evidence was presented for the protein nature of one of the serum growth factors.Partially purified from bovine serum, the factor has several marked effects upon mammalian cells in culture (appendix Al'): (a) it causes adherence of cells to a glass surface; (b) only in its presence cells assume a flattened, epithelial-like appearance; and (c) it stimulates cell multiplication.More recently, Fisher et al. ( 5), working independently with a different cell culture, HeLa Sa, have reported some of these effects.The purpose of this report is to describe the purification and properties of the protein growth factor. MATERIALS AND METHODSCulture Methods-Cells were grown on glass surfaces in a medium containing the amino acids, inorganic salts, glucose, and antibiotics suggested by Healy et al. ( 6), plus the vitamin mixture of Eagle (l), and 15 per cent bovine serum.Materials-Pooled beef serum was prepared from blood procured at a slaughterhouse.The serum was obtained by centrifugation (3") 2 to 3 hours after the blood was drawn, and it was stored at -20".No variations were noted in the activity of different batches of serum, nor was there a detectable loss in activity over a 6 month period.Human serum and serum from laboratory animals were prepared in a similar manner.Pancreatic trypsin (1: 250) and peptone (Bacto-peptone) were obtained from the Difco Laboratories, Inc., crystalline trypsin and pepsin from the Worthington Biochemical Corporation, and crystalline soy bean trypsin inhibitor from the Nutritional Biochemicals Corporation.Poly-L-lysine (about 30 residues) was a product of the Mann Research Laboratories.A sample of highly purified salminc sulfate was a gift from Dr. W. R. Carroll, pancreatic elastase (80 to 90 per cent pure) was prepared by Warner-Chilcott Laboratories, and crystalline bovine plasma albumin was obtained from the Armour Laboratories.Diethylaminoethyl-cellulose was a product of the Brown Company.