Proc. Natl. Acad. Sci. USA Vol. 73, No. 6, pp. 2023-2027, June 1976 Cell Biology Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts (human nutrition/trace element/clonal growth/fetal lung cells/Chinese hamster cells) WALLACE L. MCKEEHAN, W. GREGORY HAMILTON, AND RICHARD G. HAM Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colo. 80309 Communicated by Keith R. Porter, March 19,1976 ABSTRACT The trace element selenium is essential for text. The medium used is one of a series of media presently clonal growth of diploid fibroblasts from human fetal lung being developed specifically for diploid human cells (ref. 25, (WI-38) in media containing small amounts of serum protein. and in preparation). Maximum growth stimulation is obtained when 30 nM neu- hamster cells tralized selenious acid is added to a synthetic medium con- All growth experiments which used Chinese taining 1.5 mg/ml of dialyzed fetal bovine serum protein were carried out in medium F12 (15) containing 0.1 mM cys- (equivalent to a 3% serum concentration). Serum appears to be teine instead of the indicated 0.2 mM. a source of selenium in most culture media, since higher con- Fetal bovine serum protein (FBSP) was prepared routinely centrations of serum protein or whole serum mask the selenium as follows: 1 liter of fetal bovine serum (FBS) (Flow Laborato- requirement of WI-38 cells. Selenium is also required by a ries, was made 4.9 mM in EDTA by addition Chinese hamster cell line that can be grown in a protein-free Inglewood, Calif.) medium. of 12.5 ml of a 0.4 M stock, previously adjusted to pH 8.0 with synthetic culture 4.0 M NaOH. The solution was stirred for 15 min and dialyzed The essential role of selenium (Se) in animal nutrition is well against 40 liters of distilled water (40) for 3 days with changes established. A recent review on the biology of Se states that of the dialysis medium every 24 hr. The dialyzed protein so- Se-responsive diseases have been demonstrated in over 40 lution was centrifuged to remove insoluble material, then frozen species (1). These include liver necrosis in rats (2) and swine (3, and lyophilized to dryness. The dry powder was stored at -20° 4), exudative diathesis in chickens (5), and white muscle disease until used. Small amounts of dry powder were dissolved and, in lambs (6) and calves (7, 8). thereby, reconstituted as needed at a concentration of 50 mg/ml Advances have recently been made in the elucidation of the in a solution containing 0.01 M Tris-HCl (pH 7.3) and 0.15 M biochemical roles of Se (9). In ovine tissue, Se is incorporated NaCl. The resulting solution was equivalent in protein con- into and required for formation of a 10,000 molecular weight centration to whole serum and was used as a substitute for serum hemoprotein of muscle and heart (10, 11). Se has been positively as indicated in the text. It was sterilized by passage through identified as an integral part of the enzyme glutathione per- Millipore type GS (0.22 ,gm) filter membranes. oxidase isolated from ovine and bovine erythrocytes (12, 13). Cells and Cultures. Human Cells. Diploid human fibroblasts Despite the extensive evidence that Se is an essential nutrient (strain WI-38) from fetal lung tissue (16) at passage 14 were for many different animal species, there is a lack of direct ev- obtained from Dr. L. Hayflick (Stanford University School of idence for a requirement for Se in human nutrition (14). In this Medicine, Stanford, Calif.). The cells were grown in mass cul- paper, we present evidence that Se is required for clonal growth ture to passage 16 in Eagle's basal medium with 10% (vol/vol) of WI-38 diploid human fibroblasts. We believe this to be the FBS, transferred and grown to passages 17 through 22 in me- most direct evidence that has yet been obtained that Se may also dium F12 (15) with 10% (vol/vol) FBS. One milliliter aliquots be required by humans as an essential nutrient. Evidence is also of about 106 cells, passage 17 through 22, were frozen in liquid presented to show that an established line of Chinese hamster nitrogen after addition of 10% (vol/vol) dimethylsulfoxide to cells requires Se for clonal growth in a protein-free synthetic the medium. The frozen aliquots were used to initiate routine medium. stock cultures for use in clonal growth experiments described below. One milliliter aliquots were thawed as needed and in- MATERIALS AND METHODS oculated into Corning no. 25100 plastic tissue culture flasks (25 cm2) containing 4 ml of medium MCDB 102 (ref. 25, Table 1) Chemicals. The source of Se in all experiments was "spec- and 5 mg/ml of FBSP prepared as described above. Cultures pure" grade selenious acid from Johnson Matthey Chemicals, were grown to a confluent monolayer (2-3 days) at 370 in a Ltd., London, England, and was neutralized with 4 M NaOH. humidified 5% CO2 atmosphere. For convenience of presentation, neutralized selenious acid is Cells were harvested from the monolayer by mild trypsin referred to simply as "H2SeO3" throughout this paper. All other treatment as follows: the growth medium was removed and the chemicals used in preparation of media were from Sigma monolayer washed with two 3 ml aliquots of 0.05% (wt/vol) Chemical Co., except the inorganic salts, which were from trypsin (pH 8.0) in a solution of 120 mM NaCl, 5 mM KCl, 5.5 Fisher Scientific. mM glucose, and 26 mM NaHCO3 (Grand Island Biological Defined Media and Serum. All clonal growth experiments Co., Santa Clara, Calif.). The monolayer was exposed to a third which used WI-38 cells were carried out in medium MCDB 103 3 ml aliquot of trypsin solution preheated to 370 and monitored (Table 1) with the omission or addition of Se as indicated in the visually until the majority of cells were rounded. The trypsin solution was carefully removed and 8 ml of medium containing Abbreviations: FBSP, fetal bovine serum protein; FBS, fetal bovine 5 mg/ml of FBSP was added. The flask was shaken gently to serum; MCDB 101, 102, 103, etc., a series of media being developed for human diploid cells at the Department of Molecular, Cellular and remove cells from the culture surface, and the resulting cell Developmental Biology, University of Colorado; CHO, Chinese suspension was pipetted up and down several times to break up hamster ovary. tissue clumps. The suspension was then split 1:2 for inoculation 2023 Downloaded by guest on September 30, 2021 2024 Cell Biology: McKeehan et al. Proc. Natl. Acad. Sci. USA 73 (1976) Table 1. Composition of media MCDB 103, MCDB 102, and F12ab MCDB 103 Vitamins Inorganic salts d-Biotin 3.0 x 10-8 CaCl2 2H20 3.0 x 10 -4 Folic acid 3.0 x 10-6 CuSO4* 5H20 1.0 x 10-8 flL-ac-Lipoic acid FeSO4 7H20 3.0 x 10-6 (DL-6,8-thioctic acid) 1.0 x 10-6 - H2SeO3 3.0 x 10-8 Niacinamide 3.0 x 10 KCl 3.0 x 10-3 D-Pantothenic acid MgCl2*6H20 6.0 x 10-4 (hemi-calcium salt)C 1.0 x 10-6 NaCl 1.3 x 10-1 Pyridoxine-HCl 3.0 x 10-7 NaHCO3 1.4 x 10-2 Riboflavin 1.0 X 10-7 Na2HRO4 7H20 1.0 X 10-3 Thiamine-HCl 1.0 x 10-6 ZnSO4.7H20 5.0 x 10-7 Vitamin B12 1.O x 10-6 Amino acids Other organic compounds L-Alanine 1.0 X 10-4 Choline chloride 1.0 X 10-4 L-Arginine- HCl 1.0 X 10-3 D-Glucose 1.0 X 10-2 L-Asparagine*H20 1.0 X 10-4 Hypoxanthine 3.0 x 10-5 L-Aspartic acid 1.0 x 10-4 i-Inositol (meso-inositol) 1.0 X 10-4 L-Cysteine-HCl-H2 0 9.0 x 10-5 Linoleic acid 3.0 x 10-7 L-Glutamic acid 1.0 X 10-4 Putrescine- 2HCl 1.0 x 10-6 L-Glutamine 2.5 x 10-3 Sodium pyruvate 1.0 X 10-3 Glycine 1.0 X 10-4 Thymidine 3.0 x 10-6 L-Histidine-HCI- H20 1.O X 10-4 Buffers and indicators L-Isoleucine 3.0 x 10-5 HEPES [4-(2-hydroxyethyl)-1- L-Leucine 1.0 X 10-4 piperazineethane sulfonic acid] 2.8 x 10-2 L- Lysine- HCl 2.0 x 10 4 Phenol Red 3.3 x 10-6 L-Methionine 3.0 x 10-5 Medium MCDB 102 L-Phenylalanine 3.0 x 10-5 Same as MCDB 103 except L-Proline 3.0 x 10-4 H2SeO3 0 L-Serine 1.0 X 10-4 ZnSO4 7H20 3.0 x 10-6 L-Threonine 1.0 X 10-4 Medium F12 L-Tryptophan 1.0 x 10-5 Same as MCDB 103 except x L-Tyrosine 3.0 x 10-5 Cysteine 2.0 10-4 L-Valine 1.0 X 10-4 Glutamine 1.0 X 10-3 HEPES 0 H2SeO3 0 ZnSO4 7H20 3.0 x 10-6 a All concentrations are in moles/liter. For convenience the components of the media are listed in alphabetical order; this order does not reflect the sequence or grouping of com- ponents when preparing the media. (For detailed instructions for preparation of medium F12, from stock solutions, see Appendix A of ref.