Critical adjustment of cysteine and glutamine concentrations for improved clonal growth of WI-38 cells

Ham, Richard G.; Hammond, Susan L.; Miller, Leon L.

doi:10.1007/bf02615497

Cited by 44 publications

(13 citation statements)

References 17 publications

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“…We have not done specific experiments on oxygen toxicity. However, plating efficiencies near 50%, with no indication of toxicity, were consistently obtained in the experiments reported here and in a previous paper (25). All such experiments were performed in incubators gassed with 95% air and 5% carbon dioxide (saturated with water vapor) at an altitude of 5400 feet (1647 in).…”

supporting

confidence: 84%

“…Addition of Se to the culture medium, together with adjustment of amino acid concentrations (25), has reduced substantially the amount of serum protein needed for good clonal growth of WI-38 cells. Preliminary observations (unpublished) indicate that further reduction of the protein requirement can be achieved by supplementation of the medium with additional trace elements and by further adjustment of the concentrations of nutrients already in the medium.…”

mentioning

confidence: 99%

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Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts.

McKeehan¹,

Hamilton²,

Ham³

1976

Proc. Natl. Acad. Sci. U.S.A.

213

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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...

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supporting

confidence: 84%

mentioning

confidence: 99%

Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts.

McKeehan¹,

Hamilton²,

Ham³

1976

Proc. Natl. Acad. Sci. U.S.A.

213

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“…Thus, improvement in certain nutrient and hormonal factors in the fetal calf serum used in the current work may have corrected deficiencies that were elicited by a nutritionally inferior growth medium in the earlier study. The work of Ham and co-workers (49)(50)(51) clearly demonstrates that cellular growth requirements determined under conditions of precisely limited nutrient availability can be masked by supplementation with fetal calf serum in the concentrations used here. Indeed, the idea that in vivo aging may have occurred to a greater extent in subjects with the diabetic genotype so as to compromise the growth capacity of their cells in vitro (12,14) may just as readily be explained by auxotrophy.…”

Section: Discussionmentioning

confidence: 99%

Diabetes mellitus and genetic prediabetes. Decreased replicative capacity of cultured skin fibroblasts.

Goldstein¹,

Moerman²,

Soeldner³

et al. 1979

J. Clin. Invest.

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A B S T R A C T The idea that the gene(s) that cause diabetes mellitus can be expressed in extrapancreatic cells has been examined by tissue culture techniques. Skin biopsies were obtained from 25 normal subjects (N), 26 overt diabetics (D), 16 ofjuvenile onset (JOD) and 9 of maturity onset (MOD), and 21 subjects genetically predisposed to diabetes (P) on the basis ofmaturityonset diabetes in both parents. Each biopsy was subdivided, multiple skin fragments were explanted in vitro, and several parameters of cellular outgrowth were monitored in primary and secondary cultures until cell division ceased because of senescence. In general, the rank order of growth vigor was N > P > D although differences were often marginal and statistically significant between N and JOD and(or) MOD. Outgrowth of epithelial cells was more vigorous in N explants in early stages, but later, JOD and MOD cells grew better than those of N. Outgrowth of fibroblast cells from N explants was more vigorous both at early and later stages and required less time to achieve maximum percent outgrowth. In secondary cultures, N cells grew faster than the other three groups so that fewer days elapsed between subcultures but significant differences were only seen between N and one or two of the other groups over some of the first seven subcultures. The onset of cellular senescence occurred earlier in P and JOD cultures both in mean population doublings and calendar time. N cultures had a higher percent surviving clones after picking than MOD, and a shorter recloning time than clones ofJOD. The replicative life-spans of cultures (mean population doublings The data demonstrate that cellular growth is impaired in both JOD and MOD types of cultures and to a generally lesser extent in P cultures. This is consistent with intrinsic genetic defects but the possibility that persistent deleterious effects of in vivo pathophysiology contribute alone or in combination cannot be ruled out. Therefore, the diabetic defect(s) can be expressed in extrapancreatic cells of mesenchymal origin. This system should prove useful in exploring the interplay between genetic and environmental factors in diabetes, the mechanisms(s) of hyperglycemia and other metabolic derangements, and the propensity that affected individuals have to develop degenerative diseases.

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“…(9). Each freshly prepared batch of MCDB 102 was tested for its ability to support clonal growth of human diploid fibroblasts at between the 20th and 30th PD.…”

Section: Methodsmentioning

confidence: 99%

“…Each freshly prepared batch of MCDB 102 was tested for its ability to support clonal growth of human diploid fibroblasts at between the 20th and 30th PD. Only those batches that gave results essentially equivalent to those reported previously (9) were used. The lot of fetal bovine serum used was prescreened for its ability to promote clonal growth of WI-38 cells in Eagle's basal medium.…”

mentioning

confidence: 99%

Colony size distributions as a measure of in vivo and in vitro aging.

Smith¹,

Pereira‐Smith²,

Schneider³

1978

Proc. Natl. Acad. Sci. U.S.A.

142

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Recently it has been demonstrated that cell cultures from older human subjects, even during their first few in vitro passages, exhibit diminished replicative ability when compared to parallel cultures derived from young subjects (5). However, these studies have examined the replicative behavior of cell populations. Because human diploid cell cultures are heterogeneous in nature, it is also vital to examine the proliferative behavior of human cells as a function of aging at the level of the individual cell Recent studies on human fetal lung cells (WI-38) have indicated that the distribution of sizes of colonies derived from single cells may be an excellent indicator of the total proliferative capacity (in vitro life-span) of human diploid cell cultures (8).The studies described in this report compare the behavior of cloned human adult skin fibroblasts and fetal lung fibroblasts. In addition, the relationship between colony size distribution (CSD) and in vivo donor age is evaluated. MATERIALS AND METHODSCell cultures were established from 2-mm skin biopsies performed on nonhospitalized volunteer members of the Baltimore Longitudinal Study as described (5). Cell cultures from old (>65 years) and young (20-5 years) donors were examined after 5-15 population doublings (PDs) in vitro.The in vitro life-spans of these cell lines were determined at the Gerontology Research Center by described techniques (5). At low in vitro population doubling level (PDL), frozen stocks of these cultures were prepared by resuspending trypsinized cell suspensions in Eagle's minimal essential medium containing 10% fetal bovine serum and 5% dimethyl sulfoxide and cooled to the temperature of liquid nitrogen at a rate of '14/min.These frozen stocks of adult human skin fibroblasts were rapidly thawed in a 370 water bath, returned to tissue culture, and sent to the W. Alton Jones Cell Science Center where the colony size distributions were determined. Mass cultures were maintained in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate-buffered Eagle's basal medium with 10% fetal bovine serum (Rehatuin, Reheis Chemical Co., Kankakee, IL).All clonal growth experiments were performed in MCDB 102 which was prepared according to the procedures of Ham et al. (9). Each freshly prepared batch of MCDB 102 was tested for its ability to support clonal growth of human diploid fibroblasts at between the 20th and 30th PD. Only those batches that gave results essentially equivalent to those reported previously (9) were used. The lot of fetal bovine serum used was prescreened for its ability to promote clonal growth of WI-38 cells in Eagle's basal medium.Rapidly proliferating cultures were trypsinized prior to confluency. The cultures growing in 25-cm2 flasks were rinsed once with 5 ml of buffered medium without serum and washed with 5 ml of 0.25% crude trypsin in Eagle's basal medium. All except 0. The clonal plates were incubated undisturbed for 14 days at 370 in a 5% C02/95% air atmosphere at 98-100% relative humidity.A 2-week incubation period was ch...

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Critical adjustment of cysteine and glutamine concentrations for improved clonal growth of WI-38 cells

Cited by 44 publications

References 17 publications

Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts.

Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts.

Diabetes mellitus and genetic prediabetes. Decreased replicative capacity of cultured skin fibroblasts.

Colony size distributions as a measure of in vivo and in vitro aging.

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