The influence of culture medium volume on cell density and lifespan of human diploid fibroblasts

Ryan, Jon M.; Sharf, Barbara B.; Cristofalo, Vincent J.

doi:10.1016/0014-4827(75)90119-6

Cited by 66 publications

(20 citation statements)

References 7 publications

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“…Throughout this study optimal culture conditions were provided to enable the cells to grow and multiply at maximal rates. These conditions included provision of adequate culture medium which was changed every 24 h since the volume and frequent changing of the culture medium has been shown to affect cell density, as mentioned by Ryan et al 26 Furthermore, Hepes buffer pH 7.4, was included in both the trypsinization and culture media since pH has been shown to affect the growth pattern of cultured cells. The problem of fungal or bacterial contamination has been minimized in this study by adding streptomycin (0.1 mg ml À1 ) and penicillin (100 units ml À1 ) to the culture medium, and by standing the culture medium at room temperature (prior to the addition of fetal calf serum), for at least 1 week.…”

Section: Discussionmentioning

confidence: 99%

Changes in adenosine deaminase activity in ageing cultured human cells and the role of zinc

Ghneim

Al-Saleh

Al-Shammary

et al. 2003

Cell Biochemistry & Function

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The level of adenosine deaminase (ADA; EC 3.5.4.4) was estimated at different passages in six confluent fibroblast cultures established from forearm skin biopsies of healthy adult normal volunteers. After determination of the zinc concentration in standard growth medium, ADA activity was estimated at different passages of subculture in media with different zinc concentrations. The results indicated that the specific activity of ADA in control confluent skin fibroblast cultures (passage 2) cultivated in standard growth medium containing 15.4 microM zinc (similar to that present in normal human plasma) was equal to 226.6+/-19.64 micromol min(-1) mg(-1) protein. The results showed that there were no significant changes in ADA specific activity in any of the control cultures as the zinc concentration of the medium was increased. To characterize the passage of subculture at which fibroblasts enter the ageing phase, three marker enzymes were assayed namely, phosphofructokinase, lactate dehydrogenase and glycogen phosphorylase. The result showed that the cells enter the ageing phase at passage 20 and beyond. Further investigation showed that ADA activity of serially subcultured confluent cultures cultivated in standard growth medium significantly dropped at passages 20, 25 and 30. ADA activity however was not significantly altered in cells at passage 2, 10 and 15 cultivated in standard growth medium and in the presence of higher zinc levels (23.1, 34.6, 53.8 and 73.1 microM). Furthermore there was significant lowering of ADA activities in cells at passages 20, 25 and 30 when cells were cultured in the presence of 15.4, 23.1 and 34.6 microM zinc. Such lowered activities of ADA were restored to normal when the cells were cultured in the presence of higher zinc concentration equal to 53.8 and 73.1 microM. From the results we concluded that it is possible to restore ADA activity in aged skin fibroblasts to normal levels by raising the zinc concentration in the culture medium to four or five times the control normal plasma zinc level.

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Section: Discussionmentioning

confidence: 99%

Changes in adenosine deaminase activity in ageing cultured human cells and the role of zinc

Ghneim

Al-Saleh

Al-Shammary

et al. 2003

Cell Biochemistry & Function

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“…The volume and changing fre quency of the culture medium affects specific activities of enzymes [11], and exerts an effect on cell density [12]. The pH of the culture medium has also been shown to affect the growth pattern of cultured cells [13] and may indirectly influence the activity of several celluar enzymes [14], In addition, faulty inter pretation of certain enzyme activities can be caused by contamination of the culture with certain bacteria and mycoplasma [ 15].…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Variations Related to Glucose and Glycogen Catabolism in Serially Subcultured Human Fibroblasts

Ghneim¹

1994

Cell Physiol Biochem

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The specific activities of some glucose and glycogen degradative enzymes were studied in serially subcultured fibroblasts harvested at the same state of growth and replication. A continuous increase in the activities of phosphofmctokinase, lactate dehydrogenase, and glycogen phosphorylase was noted beyond passage 10 equalling 677 ± 74.8,170 ± 34.1, and 317 ± 57.6% of control activity at passage 25, respectively. There was a concurrent increase in the glycogen content of cells reaching a maximum of 1,498 ± 223% of control at passage 15 and maintained nearby at passages 20 and 25. Citrate synthase activity was profoundly increased at passage 15 but drastically dropped at passages 20 and 25. There were no significant variations in the above parameters between passages 1 and 10 of subculture. This enhancement of glycolytic activity which accompanies the aging (subculture) process could not be depressed as a result of improved oxygenation of the cultures.

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“…Similarly wide variations in the life-span of cultured chick cells has been reported (Hay and Strehler, 1967). While several technical factors can contribute to life-span variability (Hayflick, 1977b), the use of inadequate amounts of FBS in the growth medium and/or inoculation densities not compatible with proliferation of the entire population are the most welldocumented causes of reduced life-spans (Hayflick, 1965;Hay and Strehler, 1967;Ryan et al, 1975;Ryan, 1979a). Our results show that the use of high inoculation densities or division rate-limiting amounts of FBS in the growth medium are not compatible with proliferation of the entire nonsenescent population and as a result may be the cause of abbreviated life-spans or miss-characterization of "old" cultures.…”

Section: Resultsmentioning

confidence: 96%

The effects of division rate‐limiting amounts of fetal bovine serum on the proliferation and aging of cultured chick cells

Ryan¹,

Nielsen²,

Nelson³

1982

Journal Cellular Physiology

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Chick embryo fibroblasts serially propagated in media containing division rate-limiting amounts of fetal bovine serum underwent premature culture senescence as illustrated by accelerated declines in the number of cells incorporating 3H-thymidine, increased population doubling times, reduced cell densities at subcultivation, and reduced replicative life-spans compared to cells grown in medium containing non-rate-limiting amounts of serum. Low serum serially propagated "senescent" cultures returned to 10% serum containing medium had proliferative rates, incorporated 3H-thymidine, and attained saturation densities at confluency similar to younger cells. "Senescent" cells serially propagated in low serum and returned to 10% serum, achieved life-spans similar to cells continuously grown in the presence of 10% serum. The results of these and other studies show that cells serially propagated in the presence of division rate-limiting amounts of fetal bovine serum, or at high inoculation densities, accumulate a substantial number of cells in the population during exponential growth conditions that are not senescent but are prevented from entering DNA synthesis because of mitogen limitations. Our results indicate that the amount of serum mitogen in the growth medium affects only the rate at which cells express their genetically predetermined replicative potential and not the replicative life-span per se. These results are discussed in relation to the techniques that should be employed for studying cellular aging and the mechanism of senescent cell formation.

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The influence of culture medium volume on cell density and lifespan of human diploid fibroblasts

Cited by 66 publications

References 7 publications

Changes in adenosine deaminase activity in ageing cultured human cells and the role of zinc

Changes in adenosine deaminase activity in ageing cultured human cells and the role of zinc

Enzymatic Variations Related to Glucose and Glycogen Catabolism in Serially Subcultured Human Fibroblasts

The effects of division rate‐limiting amounts of fetal bovine serum on the proliferation and aging of cultured chick cells

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