Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein

McKeehan, Wallace L.; McKeehan, Kerstin; Hammond, Susan L.; Ham, Richard G.

doi:10.1007/bf02615100

Cited by 265 publications

(62 citation statements)

References 28 publications

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“…This organism apparently requires serum for growth ( 18), and many cultured mammalian cells require serum or serum factors for attachment or growth or both (13). One such factor, fibronectin or cold-insoluble globulin (11), apparently was not required for attachment of G. lamblia.…”

Section: Resultsmentioning

confidence: 99%

Attachment of the flagellate Giardia lamblia: role of reducing agents, serum, temperature, and ionic composition.

Gillin¹,

Reiner²

1982

Mol. Cell. Biol.

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The flagellated protozoan Giardia lamblia has been grown only in highly complex media under reduced oxygen tension. Therefore, the organic and physiological requirements for in vitro attachment and short-term (12-h) survival of this organism were determined. In defined maintenance media, a thiol reducing agent (e.g., cysteine) was absolutely required for attachment and survival of this aerotolerant anaerobe. The crude bovine serum Cohn III fraction greatly stimulated attachment and survival. Attachment was decreased at a reduced temperature (24°C as compared with 35.5°C) and absent at 12°C or below. Attachment and survival were strongly dependent upon pH and ionic strength, with optima at pH 6.85 to 7.0 and 200 to 300 mosmol/lkg. Sodium chloride was better tolerated than KCI. Reduction of Ca2+ and Mg2' to below 10-8 M did not significantly affect attachment.Giardia lamblia is an actively motile, flagellated parasitic protozoan which attaches to the human intestinal epithelium during infection (15). Giardia trophozoites attach to surfaces via the ventral adhesive or striated disk, which contains contractile proteins (5, 12;

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Section: Resultsmentioning

confidence: 99%

Attachment of the flagellate Giardia lamblia: role of reducing agents, serum, temperature, and ionic composition.

Gillin¹,

Reiner²

1982

Mol. Cell. Biol.

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“…O'Farrel et al (26) reported that 3T3 cells have a dual requirement for B12 and Hyp, whereas 3T6 cells require B12 alone. The folate or folinate requirement is related to the concentrations of adenine, dThd and glycine (22). B12 and folinate are reported to be essential when dThd or Hyp are absent (28).…”

Section: Discussionmentioning

confidence: 99%

Population-dependent requirements of vitamin B12 and metabolically related substances of several mouse cell types in serum-free, albumin-fortified medium.

Matsuya

Yamane

1986

Cell Struct. Funct.

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“…Medium MCDB 107 (23) contains 1 nM folinic acid, which facilitates the study of the effect of extracellular purines, pyrimidines, glycine, and serine (Fig. 7) on multiplication rate (20,27). t "Lipids" refer to the class I status of purified soybean phosphatidylcholine introduced in the form of vesicles or liposomes prepared by sonication (28 [S]" is the range of concentrations of individual nutrient added to deficient medium to determine the S0.50 for multiplication at different levels of FBSP between 100 and 1000 ug/ml.…”

Section: Discussionmentioning

confidence: 99%

“…Clonal multiplication was estimated by densitometric measurement of the size of colonies formed from single cells as described (4,(20)(21)(22)(23) (vol/vol) perchloric acid at 100'C. Pyruvic acid and other 2-oxocarboxylic acids were assayed as described (25).…”

Section: Introductionmentioning

confidence: 99%

“…All tissue culture methods and the development and preparation of the media series, MCDB 100, and fetal bovine serum proteins (FBSP) have been described (4,(20)(21)(22)(23). FBSP was used as a complete source of factors that stimulate cell multiplication rate above that supported by the defined medium and other optimized culture conditions (4,(20)(21)(22)(23). Multiplication rate in medium MCDB 107 is half-maximal at 43.4 + 10.2 ,gg of FBSP per ml (mean + SEM of six determinations).…”

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confidence: 99%

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Serum factors modify the cellular requirement for Ca2+, K+, Mg2+, phosphate ions, and 2-oxocarboxylic acids for multiplication of normal human fibroblasts.

McKeehan¹,

McKeehan²

1980

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

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The rate of multiplication of a population of cultured human lung fibroblasts is determined by the level of common nutrients and serum factors in the medium. By application of the principles of Henri-Michaelis-Menten kinetic analysis to cell growth in vitro, the relationship between the level of a macromolecular fraction of serum that contains growth factors and the concentration of individual nutrient that is required to support a half-maximal rate of cell multiplication was explored. The requirement for the majority of individual nutrients is independent of the level of serum factors in the medium. This includes all 20 amino acids, glucose, purines, pyrimidines, polyamines, choline, and inositol. In contrast, serum factors determine the cellular requirement for Ca2+, K+, Mg2+, Pi, and 2-oxocarboxylic acids. These nutrients are most likely involved in cellular processes related to the mechanism by which growth factors in serum control the cell multiplication rate.Serum-derived factors regulate the multiplication of untransformed cells in culture (1-4). Although much is known about the interaction of several purified growth factors with the cell membrane (5-11), less is understood about how the factors cause the biochemical and morphological events that culminate in cell division. Several studies suggest a relationship between regulatory factors and extracellular nutrients in the control of cell division (12-16). Observations on changes in uptake, intracellular pool sizes, and in rate of nutrient-related processes that occur parallel to changes in rate of DNA synthesis circumstantially implicate almost every major nutrient (15, 16). Of primary concern is which of these changes are involved in control of cell multiplication and which changes occur parallel to DNA synthesis but are not causal processes whose rates limit multiplication (17). Serum growth factors may control multiplication-limiting processes where nutrients are involved by control of the intracellular level of a nutrient around the Km for the nutrient in the rate-limiting process, or serum factors may alter the Km of the process for the nutrient whose normal level in the microenvironment is near the Km. Theoretically, both types of relationships could be reflected in an effect of serum factors on the extracellular requirement for a nutrient for multiplication of cells in culture, where extracellular levels of nutrients and serum factors can be closely controlled and manipulated. We have applied the principles that are used in classical enzyme kinetic analysis to simultaneously relate the level of serum factors and the level of individual nutrients in the medium directly to the multiplication rate of cells (18,19). The analysis revealed that serum factors modify the cellular requirement for a small subset of the nutrients commonly present in the extracellular environment. This subset consists of Ca2+, K+, Mg2+, Pi, and 2-oxocarboxylic acids. We propose that serum growth factors control cell proliferation by control of access to or requirement...

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Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein

Cited by 265 publications

References 28 publications

Attachment of the flagellate Giardia lamblia: role of reducing agents, serum, temperature, and ionic composition.

Attachment of the flagellate Giardia lamblia: role of reducing agents, serum, temperature, and ionic composition.

Population-dependent requirements of vitamin B12 and metabolically related substances of several mouse cell types in serum-free, albumin-fortified medium.

Serum factors modify the cellular requirement for Ca2+, K+, Mg2+, phosphate ions, and 2-oxocarboxylic acids for multiplication of normal human fibroblasts.

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