The trace element selenium is essential for clonal growth of diploid fibroblasts from human fetal lung in media containing small amounts of serum protein. Maximum growth stimulation is obtained when 30 nM neutralized selenious acid is added to a synthetic medium containing 1.5 mg/ml of dialyzed fetal bovine serum protein (equivalent to a 3% serum concentration). Serum appears to be a source of selenium in most culture media, since higher concentrations of serum protein or whole serum mask the selenium requirement of WI-38 cells. Selenium is also required by a Chinese hamster cell line that can be grown in a protein-free synthetic culture medium. The essential role of selenium (Se) in animal nutrition is well established. A recent review on the biology of Se states that Se-responsive diseases have been demonstrated in over 40 species (1). These include liver necrosis in rats (2) and swine (3,4), exudative diathesis in chickens (5), and white muscle disease in lambs (6) and calves (7,8).Advances have recently been made in the elucidation of the biochemical roles of Se (9). In ovine tissue, Se is incorporated into and required for formation of a 10,000 molecular weight hemoprotein of muscle and heart (10, 11). Se has been positively identified as an integral part of the enzyme glutathione peroxidase isolated from ovine and bovine erythrocytes (12,13).Despite the extensive evidence that Se is an essential nutrient for many different animal species, there is a lack of direct evidence for a requirement for Se in human nutrition (14). In this paper, we present evidence that Se is required for clonal growth of WI-38 diploid human fibroblasts. We believe this to be the most direct evidence that has yet been obtained that Se may also be required by humans as an essential nutrient. Evidence is also presented to show that an established line of Chinese hamster cells requires Se for clonal growth in a protein-free synthetic medium.MATERIALS AND METHODS Chemicals. The source of Se in all experiments was "specpure" grade selenious acid from Johnson Matthey Chemicals, Ltd., London, England, and was neutralized with 4 M NaOH. For convenience of presentation, neutralized selenious acid is referred to simply as "H2SeO3" throughout this paper. All other chemicals used in preparation of media were from Sigma Chemical Co., except the inorganic salts, which were from Fisher Scientific.Defined Media and Serum. All clonal growth experiments which used WI-38 cells were carried out in medium MCDB 103 (Table 1) Cells were harvested from the monolayer by mild trypsin treatment as follows: the growth medium was removed and the monolayer washed with two 3 ml aliquots of 0.05% (wt/vol) trypsin (pH 8.0) in a solution of 120 mM NaCl, 5 mM KCl, 5.5 mM glucose, and 26 mM NaHCO3 (Grand Island Biological Co., Santa Clara, Calif.). The monolayer was exposed to a third 3 ml aliquot of trypsin solution preheated to 370 and monitored visually until the majority of cells were rounded. The trypsin solution was carefully removed and 8 ml of medi...

A chemically defined, protein-free medium (designated CFBI 1000, where CFBI = Clayton Foundation Biochemical Institute) that supports human peripheral lymphocyte proliferation has been developed. This medium allows exploration of individual metabolic differences by varying the medium composition as well as providing a base to explore further the mechanisms of lymphocyte activation in a system initially free of added macromolecular species other than mitogen. The peripheral blood lymphocyte is an ideal system for metabolic studies because it is easily obtained, is a primary resting cell that can be activated to proliferate, and presumably reflects both the genetic makeup and biochemical environmental history of the individual at the time the cells were formed.

A protein-free synthetic medium, MCDB 301, has been developed for clonal growth of Chinese hamster ovary cell lines. Medium F12 was developed originally for that purpose, but later failed to support good growth without small amounts of serum protein. Growth was restored by the addition of nonphysiological amounts of commercially prepared thyroxine or smaller amounts of the trace element selenium. The thyroxine preparation was shown to contain sufficient selenium to account for all of its growth-promoting activity. MCDB 301 contains increased concentrations of calcium chloride and glutamine, and a smaller amount of cysteine than medium F12. It also has been supplemented with 19 inorganic ions, in addition to selenium and those in medium F12, in order to insure against possible future deficiencies as chemicals are purified further. A Chinese hamster lung line which will not grow in MCDB 301 alone will grow when the medium is supplemented either with methylcellulose or with insulin. The growth-promoting activity is thought to be an impurity shared in common by both substances. The probable "essential" role of impurities in cellular growth in most synthetic media and the problems involved in attempting to develop a truly "defined" medium are discussed.