The Effect of Medium Changes on the Growth and Metabolism of the Human Diploid Cell, W1-38

1971 ◽  
Vol 8 (1) ◽  
pp. 43-52
Author(s):  
J. B. GRIFFITHS
Keyword(s):  
Protein Synthesis ◽  
Contact Inhibition ◽  
Cell Yield ◽  
Unexpected Result ◽  
Inhibitory Interaction ◽  
Inhibition Of Growth ◽  
Rna And Protein Synthesis ◽  
Medium Change ◽  
Human Diploid Cells ◽  
Diploid Cells

It has been established that although an inhibitory interaction occurs when a culture of human diploid cells become crowded together (contact inhibition of growth), multiple-layered cell sheets are obtained by using a continuous medium perfusion culture. A similar effect is obtained when the culture medium is changed at frequent intervals, and this paper reports the effects of a medium change on cell growth and metabolism. A direct relationship was found between cell yield and the number of medium changes given to a culture. This was an unexpected result because normally when a culture is prolonged by additional feeding the cell yield shows a diminishing return. The amino acid and glucose uptakes and growth yields (the ratio of the amount of cell dry weight produced to substrate used) were determined and they also showed that a unit amount of growth occurred per medium change, and that cessation of growth was accompanied by cessation of nutrient uptake and metabolism. Medium changes had a profound affect on cellular metabolism, especially on DNA and protein synthesis. As a culture approached confluency, DNA, RNA and protein synthesis were sequentially inhibited. After a medium change there was a sequential stimulation of DNA, RNA and protein synthesis in the same order as they were inhibited. The inhibitory mechanism that is affected by cell crowding is obviously reversed by a medium change. The results presented in this paper suggest that contact inhibition of growth primarily affects DNA synthesis and that if the cell is able to take up a sufficient supply of nutrients in a crowded culture then this inhibition can be overcome.

Growth of human diploid cells (strain MRC-5) in defined medium; replacement of serum by a fraction of serum ultrafiltrate

1979 ◽  
Vol 35 (1) ◽  
pp. 381-392
Author(s):  
K. Lambert ◽  
S.J. Pirt
Keyword(s):  
Amino Acids ◽  
Active Material ◽  
Maximum Yield ◽  
Calf Serum ◽  
Defined Medium ◽  
Cell Yield ◽  
Maximum Cell ◽  
Macromolecular Component ◽  
Human Diploid Cells ◽  
Diploid Cells

A calf serum ultrafiltrate fraction permitted growth for at least 3.5 generations, including one subculture, of MRC-5 cells in defined medium in the absence of whole serum. The active material has a molecular weight of 10 000 Daltons or less. This suggests that there may be no requirement for a large macromolecular component of serum. The ultrafiltrate was assayed by maximum cell yield from a serum-limited inoculum in a defined medium containing non-limiting amounts of vitamins, amino acids, glucose, a 68-component supplement, iron and methylcellulose. The levels of vitamins, amino acids and glucose were based on quantitative measurements of uptake and the levels of the other components by minimum amount required for maximum yield in defined medium without ultrafiltrate or serum. With excess ultrafiltrate maximum cell yield was limited by the defined part of the medium, probably the supplement. The cell doubling time in defined medium with ultrafiltrate fractions was 70 h compared with 27 h in the medium with serum. Excess ultrafiltrate did not inhibit growth. The lowered growth rate is attributed to a nutritional deficiency in the supplement.

The Effect of Cell Population Density on Nutrient Uptake and Cell Metabolism: a Comparative Study of Human Diploid and Heteroploid Cell Lines

1972 ◽  
Vol 10 (2) ◽  
pp. 515-524
Author(s):  
J. B. GRIFFITHS
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Dna Synthesis ◽  
Nutrient Uptake ◽  
Cell Lines ◽  
Acid Concentration ◽  
Contact Inhibition ◽  
Inhibition Of Growth ◽  
Extracellular Amino Acid

The possibility that contact inhibition of growth in cultures of human diploid cells is influenced by the effects of cell crowding on nutrient uptake by the cells was investigated. Two human lung cell lines were compared, the diploid line MRC-5 and the heteroploid line L-132. In pre-confluent cultures the ability of these 2 cell types to accumulate amino acids was very similar. Post-confluent L-132 cells showed very little change from the pre-confluent cultures but the ability of MRC-5 cells in post-confluent cultures was greatly reduced. The intracellular concentrations of various amino acids necessary to achieve the maximum rate of protein synthesis were found. These values were identical for sparse and crowded cultures but due to the reduced uptake ability of crowded MRC-5 cells a far higher external amino acid concentration was required in post-confluent cultures. This meant that although amino acids did not become growth-limiting until over 80% utilized in pre-confluent cultures, in post-confluent cultures they became growth-limiting when only 50% utilized. Although protein synthesis was significantly affected by extracellular amino acid concentration and cell crowding, thus contributing towards the effect of contact inhibition of growth, DNA synthesis was shown to be the major metabolic function in contact inhibition. Increased cell density had a very inhibitory effect on DNA synthesis in MRC-5 cultures, but not in L-132 cultures, and this was unaffected by extracellular amino acid and glucose concentration.

scholarly journals Protein, nucleic acid and starch metabolism in the duckweed Spirodela oligorrhiza, treated with cytokinins

1972 ◽  
Vol 129 (2) ◽  
pp. 403-417 ◽  
Author(s):  
P. J. A. McCombs ◽  
R. K. Ralph
Keyword(s):  
Protein Synthesis ◽  
Specific Protein ◽  
Starch Metabolism ◽  
Animal Cells ◽  
Stringent Control ◽  
Inhibition Of Growth ◽  
Rna And Protein Synthesis ◽  
Membrane Bound ◽  
Spirodela Oligorrhiza ◽  
Protein Nucleic Acid

Bacteria-free cultures of Spirodela oligorrhiza continue to increase in frond number for 2 to 3 days after transfer to darkness. There is then no further increase in frond number for 3 to 4 weeks, although DNA, RNA and protein synthesis continue at decreased rates and starch accumulates in the plants. We refer to such ‘non-growing’ plants in darkness as dormant. Adding kinetin to dormant Spirodela initiated increased DNA, RNA and protein synthesis within 1h, although new fronds were not detected until 24h after the addition of kinetin. The frond number then continued to increase. Starch accumulated in dormant plants. Accumulation of starch appeared to be a consequence of inhibition of growth rather than the converse. No evidence was obtained for a block in [14C]glucose metabolism that might explain the lack of growth in darkness in the absence of kinetin. In darkness, more ribosomes were membrane-bound in dormant Spirodela than in Spirodela growing with kinetin. Similarities between the response of Spirodela to darkness, stringent control in bacteria and pleiotypic controls in animal cells are discussed. It is suggested that all three processes are ultimately controlled by specific protein kinases that are individually sensitive to different effectors.

The Quantitative Utilization of Amino Acids and Glucose and Contact Inhibition of Growth in Cultures of the Human Diploid Cell, WI-38

1970 ◽  
Vol 6 (3) ◽  
pp. 739-749
Author(s):  
J. B. GRIFFITHS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Basal Medium ◽  
Diploid Cell ◽  
Contact Inhibition ◽  
Growth Media ◽  
Acid Uptake ◽  
Human Diploid Cell ◽  
Inhibition Of Growth ◽  
Diploid Cells

There are many reports in the literature showing that contact inhibition of growth is affected by the culture medium. A quantitative study of amino acid and glucose uptake by the human diploid cell line, WI-38 was carried out to determine more precisely what effect nutritional factors have on contact inhibition of growth. Eagle's minimal essential medium (MEM) was found to support higher cell yields than Eagle's basal medium (BME) and for growth to continue beyond 96 h a medium change was essential. However, analysis of the used growth media showed that neither amino acids nor glucose were fully depleted after 96 h. The rate of glucose utilization was in the range 65-100µg/mg dry wt./h and this agreed very closely with the results of other authors. The pattern of amino acid uptake also closely resembled that for other cell lines except that the utilization of cystine was higher. The nutritional requirements were further studied as the results from the medium analyses failed to explain the growth-promoting activity of MEM. Daily medium changes greatly increased cell yields even though the medium nutrients were not exhausted. This effect was dependent upon fresh medium being used and the only medium component found to be of importance was the amino acid complement. These results are discussed in relation to the low saturation density of diploid cells in culture and a possible explanation is proposed in terms of differences in the cell membrane between normal and altered cells.

The Effects of Adapting Human Diploid Cells to Grow in Glutamic Acid Media on Cell Morphology, Growth and Metabolism

1973 ◽  
Vol 12 (2) ◽  
pp. 617-629 ◽  
Author(s):  
J. B. GRIFFITHS
Keyword(s):  
Glutamic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Uptake ◽  
Transformed Cells ◽  
Acid Uptake ◽  
Acid Media ◽  
Inhibition Of Growth ◽  
Dependent Inhibition ◽  
Human Diploid Cells ◽  
Diploid Cells

Two lines of human diploid cells, the W1-38 and MRC-5, were adapted to utilize glutamic acid in place of glutamine. This adaptation resulted in (a) more cells per unit culture area, (b) an alteration in cell size and protein content, (c) a morphological change of the cells from fibroblastic to epithelial-like, and (d) increased metabolic activity. Changes in the agglutinability of adapted cells by 3 lectins together with the results from polyacrylamide gel electrophoresis could be interpreted as a change in plasma membrane structure. Comparative studies of unadapted, adapted and transformed cells showed that adaptation to glutamic acid produced cells with a metabolism and amino acid uptake similar to those of transformed cells. These changes were reversible and were not accompanied by any apparent karyological change. The significance of these results for the study of density-dependent inhibition of growth is discussed.

scholarly journals Evidence for endogenous polypeptide-mediated inhibition of cell-cycle transit in human diploid cells.

1982 ◽  
Vol 94 (1) ◽  
pp. 187-192 ◽  
Author(s):  
G C Burmer ◽  
C J Zeigler ◽  
T H Norwood
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Dna Synthesis ◽  
Tritiated Thymidine ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Human Diploid Fibroblasts ◽  
Senescent Phenotype ◽  
Human Diploid Cells ◽  
Diploid Cells

Previous studies have shown that the senescent phenotype is dominant with respect to DNA synthesis in fusions between late passage and actively replicating human diploid fibroblasts. Brief postfusion treatments with the protein synthesis inhibitor cycloheximide (CHX) or puromycin have been found to significantly delay (by 24-48 h) the inhibition of entry into DNA synthesis of young nuclei in heterokaryons after fusion with senescent cells. A significant fraction of the senescent nuclei incorporated tritiated thymidine in CHX-treated heterokaryons. The optimal duration of exposure to CHX was 1-3 h immediately after fusion, although treatments beginning as late as 9 h after fusion elevated the heterokaryon labeling index. Prefusion treatments with CHX were without a significant effect. These results are consistent with the interpretation that regulatory cell cycle inhibitor(s) which are dependent upon protein synthesis may be present in heterokaryons between senescent and actively replicating cells.

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