Effect of the growth conditions on the expression of cell-surface-associated platelet-derived growth factor receptors in mouse fibroblasts

The conditions affecting the appearance and disappearance of platelet-derived growth factor (PDGF) receptors from the pool of active cell surface-associated receptors were studied. Receptor molecules were revealed in intact Swiss 3T3 fibroblasts by their ability to bind 125I-labeled PDGF and, due to their property to become phosphorylated in tyrosine following ligand binding, by antibodies to phosphotyrosine. PDGF receptor molecules were found to be quite scarce in exponentially growing fibroblasts as compared to quiescent cells. When growing cells were either shifted to a medium containing plasma or received suramin in the culture medium, cell surface-associated PDGF receptors largely increased. This process required about 12 h. Incubation of quiescent cells in serum, but not in plasma, induced a slow decrement of ligand-activatable receptors. In the presence of PDGF the rate of receptor removal from the cell surface was very rapid and was a function of the PDGF concentration. Quiescent cells deprived of cell-surface receptors by incubation with PDGF reexpressed PDGF receptors in about 14 h.     

Effect of serum step-down on protein metabolism and proliferation kinetics of NHIK 3025 cells

Human NHIK 3025 cells growing exponentially in 30% or 3% serum had population doubling times of 19.1 and 27.6 hours, respectively. These values were equal to the calculated protein doubling times (17.6 and 26.5 hours, respectively), showing that the cells were in balanced growth at both serum concentrations. Stepdown from 30% to 3% serum reduced the rate of protein synthesis within 1–2 hours, from 5.7% hour to 4.3% hour, while the rate of protein degradation was unchanged (1.7%/hour). In cells synchronized by mitotic selection from an exponentially growing population, the median cell cycle durations in 30% and 3% serum were 17.2 and 23.6 hours, respectively, which were also in good agreement with the protein doubling times. The median G₁ durations were 7.1 and 9.6 hours, respectively. Thus the duration of G₁ relative to the total cell cycle duration was the same in the two cases. Complete removal of serum for a period of 3 hours resulted in a 3-hour prolongation of the cell cycle regardless of the time after mitotic selection at which the serum was removed. For synchronized cells, the rate of entry into both the S phase and into the subsequent cell cycle were reduced in 3% serum as compared to 30% serum, the former rate being significantly greater than the latter at both serum concentrations. Our results thus indicate that these cells are continuously dependent upon serum throughout the entire cell cycle.     

The relationship between yeast cell size and cell division in Candida albicans

The mean size and percentage of budded and unbudded cells of Candida albicans grown in batch culture over a wide range of doubling times have been measured. Cell volume decreased with increased doubling time and a nonlinear approach to an asymptotic minimum was observed. When cells were separated by age according to bud scars, each age showed a similar decrease. During each cell division cycle, size increased slowly during both budded and unbudded periods so that each generation was significantly larger than the preceding. There was no difference in size between the parent portion of budded cells and unbudded cells of the same age. Time-lapse photomicroscopy of cells growing on solid medium showed that cells divide asymmetrically with larger parents having a shorter subsequent cycle time than the smaller daughter, although the time utilized for bud formation was similar. When cells were shifted from a medium supporting a low growth rate and small size to a medium supporting a faster growth rate and larger size, both budded and unbudded cells increased significantly in size. As the doubling time increased, both the budded and unbudded portions of parental and daughter cycles increased.     

Intracellular protein degradation in serum-deprived human fibroblasts.

IMR90 human fibroblasts were labelled by incubation of cells for 48 h in medium containing 10% serum and [3H]leucine. The labelled protein was degraded at a rate of 1%/h during a subsequent incubation in medium with 10% serum. Incubation in medium without serum caused a transient enhancement of the degradation of endogenous protein, which was also found in cells labelled in medium without serum. The degradation of micro-injected haemoglobin was enhanced by serum deprivation in a non-transient manner. These results suggest that enhanced degradation in serum-free medium occurs only for a subpopulation of cell proteins and that it appears transient because the major part of the pool of susceptible endogenous proteins is being degraded during the first 20-30 h in serum-free unlabelled medium. Protein turnover in various cell compartments was measured by a double-labelling technique. Most of the enhanced degradation in serum-deprived cultures (73-83%) was due to breakdown of cytosolic proteins. The enhanced degradation of cytosolic proteins seemed to affect several proteins irrespective of their molecular mass or metabolic stability.     

The role of increased proteolysis in the atrophy and arrest of proliferation in serum-deprived fibroblasts

When cultured fibroblasts are deprived of serum, the degradation of long-lived proteins and RNA increases, the cells stop proliferating, and they decrease in size. To determine the role of the increased protein catabolism in these responses, we studied the effects of inhibitors of intralysosomal proteolysis in Balb/c 3T3 cells. When these cells were placed in serum-deficient medium (0.5% serum), the rate of degradation of long-lived proteins increased about twofold within 30 min. This increase was reduced by 50-70% with inhibitors of lysosomal thiol proteases (Ep475 and leupeptin) or agents that raise intralysosomal pH (chloroquine and NH4Cl). By contrast, these compounds had little or no effect on protein degradation in cells growing in 10% serum. Thus, in accord with prior studies, lysosomes appear to be the site of the increased proteolysis after serum deprivation. When 3T3 cells were deprived of serum for 24-48 hours, the rate of protein synthesis and the content of protein and RNA and cell volume decreased two- to fourfold. The protease inhibitor, Ep475, reduced this decrease in the rate of protein synthesis and the loss of cell protein and RNA. Cells deprived of serum and treated with Ep475 for 24-48 hours had about twice the rate of protein synthesis and two- to fourfold higher levels of protein and RNA than control cells deprived of serum. The Ep475-treated cells were also about 30% larger than the untreated cells. Thus, the protease-inhibitor prevented much of the atrophy induced by serum deprivation. The serum-deprived fibroblasts also stopped proliferating and accumulated in the G1 phase of the cell cycle. The cells treated with Ep475 accumulated in G1 in a manner identical to untreated serum-deprived cells. Other agents which inhibited protein breakdown in serum-deprived cells also did not prevent the arrest of cell proliferation. Thus the enhancement of proteolysis during serum deprivation appears necessary for the decrease in size and protein synthesis, but probably not for the cessation of cell proliferation. When cells deprived of serum in the presence or absence of Ep475 were stimulated to proliferate by the readdition of serum, the larger Ep475-treated cells began DNA synthesis 1-2 hours later than the smaller untreated cells. Thus, after treatment with Ep475, the rate of cell cycle transit following serum stimulation was not proportional to the cell's size, protein, or RNA content, or rate of protein synthesis.     

Cytochalasin B releases a major surface-associated glycoprotein, fibronectin, from cultured fibroblasts

BHK21 cells cultured in minimal essential medium (Eagle) supplemented with 10% dialyzed fetal calf serum did not grow as they did in whole serum containing medium. Logarithmic growth was, however, initiated after a lag period, the length of which was dependent upon the cell density: medium volume ratio. The quiescent cells conditioned the medium during this lag period, and growth stimulation was apparently due to the release of serine into the medium. Cells cultured in 10% dialyzed serum plus the low molecular weight fraction of serum (serum dialysate), grew with kinetics similar to cells cultured in serum containing medium. When serum dialysate was chromatographed on Bio-gel P-2 the growth promoting activity eluted with the amino acids. Each of the non-essential amino acids was tested for its ability to stimulate the growth of cells in 10% dialyzed serum. Serine was capable of stimulating cell growth to the same extent as 10% serum dialysate and its concentration optimum was similar to its concentration in 10% serum dialysate. The remaining non-essential amino acids were either slightly stimulatory or had no effect on cell growth. Shifting a logarithmically growing population of cells to serine-free medium resulted in the accumulation of 95% of the cells in the G1 phase of the cell cycle within 24 h. Escape from the G1 block could occur if serine was added to the medium or if the cells were allowed to condition the medium. Entry of cells into S phase after the addition of 0.05 μmoles/ml of serine followed a 4–6 h lag and 80% of the cells were synthesizing DNA 12 h after shift-up.     

Effect of conditioned medium factors on productivity and cell physiology in Trichoplusia ni insect cell cultures

The influence of conditioned medium (CM) on cell physiology and recombinant protein production in Trichoplusia ni insect cells (T. ni, BTI-Tn-5B1-4) has been investigated. Cell cycle analysis showed that a high proportion of the cell population (80-90%) was in G1 during the whole culture, indicating that the S and G2/M phases are short relative to the G1 phase. Directly after inoculation, a rapid decrease of the S-phase population occurred, which could be observed as a lag-phase. The following increase in the number of cells in S occurred after 7 h of culture for cells in fresh medium, whereas for cells with the addition of CM it occurred at an earlier time point (5 h) and these cells had therefore a shorter lag-phase. The initial changes in the S-phase population were also affected by the inoculum cell density, as higher seeding cell densities resulted in a more rapid increase in the S-phase population after inoculation. These changes in cell cycle distribution were reflected in the cell size, and the CM-cells were smaller than the cells in fresh medium. Recombinant protein production in T. ni cells was improved by the addition of CM. The specific productivity was increased by 30% compared to cells in fresh medium. This beneficial effect was seen between 20 and 72 h of culture. In contrast, the highest specific productivity was obtained already at 7 h for the cells in fresh medium and then decreased rapidly. The total product concentration was around 30% higher in the culture with CM compared to the culture in fresh medium, and the maximum product concentration was obtained on day 2 compared to day 3 for the cells in fresh medium. Our results indicate that the positive effect on productivity by CM is related to its growth-promoting effect, suggesting that the proliferation potential of the culture determines the productivity.     

Effects of cell density and cell growth alterations on cyclic nucleotide levels in cultured human diploid fibroblasts.

cGMP and cAMP concentrations were studied in cultures of two strains of normal human diploid lung fibroblasts, WI38 and KL-2, under various conditions which alter growth rate. Higher levels of cAMP were found in fibroblasts grown in medium with low (0.1 – 1.0%) serum concentration and thus exhibiting a decreased rate of growth. A rise in cAMP also preceded the decreased growth rate when medium was not changed for 4 days or longer (starvation). The reinitiation of cell growth by addition of fresh medium containing the standard 10% serum to either starved or serum-restricted cells was preceded by a rapid drop in cAMP level. Cellular cAMP levels increased to a moderate extent as sparse cultures first increased in density, but did not continue to rise as the culture approached saturation density. cGMP levels were inversely related to cell density: much higher cellular cGMP levels were found at low density than at higher cell density, whether cells were rapidly proliferating under standard growth conditions or had their growth arrested by omission of medium change or restriction of serum. Thus, under these conditions the steady state levels of cGMP appear to be related to cell density rather than rate of cell proliferation. However, a transient but appreciable increase in cGMP did occur upon the addition of fresh medium containing 10% serum to starved or serum-restricted cells, a condition leading to reinitiation of cell proliferation. Smaller but significant increases in cGMP were also evident following routine addition of fresh medium with serum to growing cells fed every other day and following mild EDTA-trypsin treatment of confluent WI38 fibroblasts. Thus, at least dual control mechanisms appear to be involved in the regulation of cGMP levels. Comparison of mid- and late-passage WI38 cells revealed no significant differences either in the levels of cGMP at sparse densities or in the density-dependent change in levels. These results suggest that levels of both cAMP and cGMP are influenced by cell density and also by conditions which alter the rate of cell proliferation.     

Kinetic aspects of Ki-67 antigen expression in a normal cell line

Using a normal cell line derived from a human fetus, the disappearance and reappearance of the Ki-67-reactive antigen following modification of the cell cycle was observed and estimated immunohistologically. It was found that G1/G0 arrest induced by serum deprivation resulted in loss of the antigen in 24 h in all but a few (usually less than 10%) of cells. Return to normal medium and resumption of growth was accompanied by reappearance in 30 h. When entry into S-phase was prevented by desferrioxamine, reappearance of the antigen still occurred but only lasted for about 24 h. Inhibition of protein synthesis with cycloheximide also caused fading and eventual loss of immunostaining. In view of the ease with which this antigen becomes undetectable with cessation of protein synthesis and interruption of the cell cycle, we agree with those who advise caution in the use of Ki-67 to measure growth fraction in changeable cell populations such as tumours.     

Effect of particle loading on GM-CSF production by Saccharomyces cerevisiae in a three-phase fluidized bed bioreactor

Continuous production of a recombinant murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) by immobilized yeast cells, Saccharomyces cerevisiae strain XV2181 (a/a, Trp1) containing plasmid palphaADH2, in a fluidized bed bioreactor was studied at a 0.03 h(-1) dilution rate and various particle loading rates ranging from 5% to 33% (v/v). Cells were immobilized on porous glass beads fluidized in an air-lift draft tube bioreactor. A selective medium containing glucose was used to start up the reactor. After reaching a stable cell concentration, the reactor feed was switched to a rich, nonselective medium containing ethanol as the carbon source for GM-CSF production. GM-CSF production increased initially and then dropped gradually to a stable level. During the same period, the fraction of plasmid-carrying cells declined continuously to a lower level, depending on the particle loading. The relatively stable GM-CSF production, despite the large decline in the fraction of plasmid-carrying cells, was attributed to cell immobilization. As the particle loading rate increased, the plasmid stability also increased. Also, as the particle loading increased from 5% to 33%, total cell density in the bioreactor increased from 16 to 36 g/L, and reactor volumetric productivity increased from 0.36 to 1.31 mg/L.h. However, the specific productivity of plasmid-carrying cells decreased from 0.55 to 0.07 mg/L.g cell. The decreased specific productivity at higher particle loading rates was attributed to reduced growth efficiency caused by nutrient limitations at higher cell densities. Both the reactor productivity and specific cell productivity increased by two- to threefold or higher when the dilution rate was increased from 0.03 to 0.07 h(-1). (c) 1996 John Wiley & Sons, Inc.     

Assembly of the sarcoplasmic reticulum. Biosynthesis of calsequestrin in rat skeletal muscle cell cultures.

Temporal patterns of biosynthesis of the sarcoplasmic reticulum protein, calsequestrin, were analyzed and compared with rates of ATPase synthesis in primary cultures of rat skeletal muscle cells. Rates of synthesis were measured by the incorporation of radioactive leucine into the isolated proteins. Cells at various stages of differentiation were incubated for 2 h with tritium-labeled leucine and extracted with detergent. The extracts were incubated with antibodies specific against calsequestrin or the ATPase and immunoprecipitates were separated by disc gel electrophoresis. Incorporation of radioactivity into bands identified as calsequestrin or the ATPase was analyzed by counting of gel slices. In Dulbecco's modified Eagles medium (DME medium) containing 0.1 volume of horse serum and 0.005 volume of chick embryo extract, the cells began to fuse after about 50 h in culture, forming multinucleated myotubes. Calsequestrin synthesis was barely detectable after 24 h in culture. After 44 h, before fusion of myoblasts began, the rate of calsequestrin synthesis increased severalfold. The rate of synthesis continued to increase until about 72 h and then diminished. If cells were transferred at 44 h to DME medium containing 0.2 volume of fetal calf serum and 0.08 volume of chick embryo extract, fusion was delayed by about 20 h. In this medium the rate of calsequestrin synthesis diminished after a peak at 44 h but, by contrast, the rate of synthesis of the ATPase increased dramatically following fusion at about 80 h. If cells were transferred at about 40 h to DME medium containing 0.1 volume of horse serum and only 60 muM Ca2+ the cells did not fuse and, again, the rate of calsequestrin synthesis was diminished after a peak at about 40 h. By contrast the rate of ATPase synthesis increased sharply in spite of the lack of fusion. Both proteins were degraded with a half-life of about 20 h. These studies show that the synthesis of calsequestrin, an extrinsic membrane protein, and the ATPase, an intrinsic protein of the same membrane, are synthesized under separate control.     

Changes in the uptake of 2-deoxy-D-glucose in BALB-3T3 cells chemically transformed in culture

Balb/3T3 cells transformed in culture by chemical carcinogens were shown to multiply in a medium supplemented with 2% calf serum or with 10% agamma new-born calf serum. The cell lines that multiply well in medium supplemented with 10% agamma serum produced a higher incidence of tumors in X-irradiated weanling mice than the lines that multiply poorly. The difference in 2-deoxy-D-glucose uptake into exponentially growing transformed and un-transformed cells was 50–100%. In crowded cultures untransformed Balb/3T3 cells ceased taking up the sugar, while chemically transformed cells continued at the same rate even at high cell densities; thus, the difference became greater in crowded cultures. When the serum concentration in the media was reduced from 10% to 2%, untransformed Balb/3T3 cells took up the sugar at a reduced rate, while chemically transformed cells were only slightly affected; agamma new born calf serum supplemented medium had no effect on sugar uptake in any of the cells. When the serum concentration was changed from 2% to 10%, untransformed cells increased sugar uptake followed by cell division. The immediacy (within 15 min) of the response in the sugar uptake to 10% serum concentration suggested that the increased uptake rate and the consequent higher concentration of the sugar (D-glucose in normal situation) within Balb/3T3 cells triggered the cell cycle. Chemical carcinogens appear to alter permanently the uptake mechanism for a key nutrient.     

Regulation of ribosomal protein mRNA content and translation in growth-stimulated mouse fibroblasts

When resting (G₀) mouse 3T6 fibroblasts are serum stimulated to reenter the cell cycle, the rates of synthesis of rRNA and ribosomal proteins increase, resulting in an increase in ribosome content beginning about 6 h after stimulation. In this study, we monitored the content, metabolism, and translation of ribosomal protein mRNA (rp mRNA) in resting, exponentially growing, and serum-stimulated 3T6 cells. Cloned cDNAs for seven rp mRNAs were used in DNA-excess filter hybridization studies to assay rp mRNA. We found that about 85% of rp mRNA is polyadenylated under all growth conditions. The rate of labeling of rp mRNA relative to total polyadenylated mRNA changed very little after stimulation. The half-life of rp mRNA was about 11 h in resting cells and about 8 h in exponentially growing cells, values which are similar to the half-lives of total mRNA in resting and growing cells (about 9 h). The content of rp mRNA relative to total mRNA was about the same in resting and growing 3T6 cells. Furthermore, the total amount of rp mRNA did not begin to increase until about 6 h after stimulation. Since an increase in rp mRNA content did not appear to be responsible for the increase in ribosomal protein synthesis, we determined the efficiency of translation of rp mRNA under different conditions. We found that about 85% of pulse-labeled rp mRNA was associated with polysomes in exponentially growing cells. In resting cells, however, only about half was associated with polysomes, and about 30% was found in the monosomal fraction. The distribution shifted to that found in growing cells within 3 h after serum stimulation. Similar results were obtained when cells were labeled for 10.5 h. About 70% of total polyadenylated mRNA was in the polysome fraction in all growth states regardless of labeling time, indicating that the shift in mRNA distribution was species specific. These results indicate that the content and metabolism of rp mRNA do not change significantly after growth stimulation. The rate of ribosomal protein synthesis appears to be controlled during the resting-growing transition by an alteration of the efficiency of translation of rp mRNA, possibly at the level of protein synthesis initiation.     

The origin of variability in cell cycle durations of NHIK 3025 cells

The origin of cell cycle variability was investigated in NHIK 3025 cells synchronized by mitotic selection from an exponentially growing population. The variability in G1 durations was measured by flow cytometric analysis of the fraction of cells in G1 as a function of time after mitotic selection. Immediately before the first cells entered S, medium containing 2.0 mM thymidine was added to the cells, and removed when all the cells had reached S. Since the cells had approximately the same DNA content upon removal of the thymidine, the variability in the durations of S+G2+M was measured by counting the fraction of undivided cells as a function of time after removing the thymidine. Such a thymidine treatment did not affect the naturally occurring variability in cell cycle durations generated after the start of S. The results indicate that the cell cycle variability of NHIK 3025 cells can be adequately described by a cell cycle model consisting of at least two compartments, which the cells leave according to first order kinetics. The model accounts for the initial shoulder of the curve representing the fraction of undivided cells as a function of time after mitotic selection. Furthermore, it accounts for the reduction in the rate of entry into the subsequent cell cycle compared to the rate of entry into S. Both rate constants were equally reduced after serum stepdown.     

Glycolipid membrane anchored recombinant protein production from CHO cells cultGred on porous microcarriers

Recombinant proteins were harvested from Chinese hamster ovary (CHO) cells by a controlled release process, which increased the purity and concentration of the harvested protein. Recombinant human melano-transferrin (p97) was expressed linked to the outer surface of CHO cells by a glycosyl-phosphatidylinositol (GPI) membrane anchor. Cells were grown to confluence in T-flask culture, and the p97 harvested by replacing the growth medium for 30 min with phosphate-buffered saline (PBS) containing 10 mU/mL phosphatidylinositol-phospholipase C (PI-PLC). The GPI anchor was selectively cleaved by PI-PLC. In fresh medium, the CHO cells regained over 95% of their p97 expression within 40 h. The process was repeated for eight harvests. Harvested protein concentrations varied from 1.5 to 3.8 mug/mL due to difficulties in maintaining stable confluent T-flask cultures. Harvesting from cells growing on porous microcarriers was investigated to increase p97 product concentrations and to overcome culture stability problems. Semicontinuous cultures were maintained in spinners for up to 76 days with average bioreactor cell densities of over 10(7) cell/mL. The p97 was harvested at up to 100 mug/mL and 30% purity with protein production remaining stable for 4 harvest cycles. Production of high levels of p97 from CHO cells was maintained at 0.5% serum. (c) 1994 John Wiley & Sons, Inc.     

Calcitonin-responsive clonal cell line from porcine kidney (PK(15)) in serum-free medium

PK(15), a homogeneous epithelial cell line from porcine kidney which was originally established through single cell cloning from PK-2a, was found to respond to [Asu1,7]eel calcitonin with an increase in adenosine 3':5'-cyclic monophosphate (cAMP) content, but do not respond to parathyroid hormone or arginine vasopressin. These cells were able to grow in a synthetic medium (a 1:1 mixture of Dulbecco's modified Eagle's MEM and Ham's F12 medium) without any supplementary factor. The medium supplemented with selenous acid, transferrin, and insulin permitted a growth rate equivalent to those in serum containing medium. When grown in the serum-free defined medium, these cells showed an increase in cAMP content in response to [Asu1,7]eel calcitonin to approximately the same degree as in the serum containing medium (10% fetal calf serum). Our present study first indicates that PK(15) cells are capable of growing in the serum-free defined medium retaining the calcitonin responsiveness of the original cells.     

Effect of different fetal bovine serum concentrations on the replicative life span of cultured chick cells

The effect of Eagle's minimal essential medium, containing different fetal bovine serum (FBS) concentrations, on the proliferation and replicative life span of cultured chick cells has been studied. Our results showed that the rate of chick cell proliferation and the cell density at stationary phase increased as a function of serum concentration between 5 and 30% FBS. The replicative life span of cultured chick cells was dependent on the FBS concentration between 5 and 20% in a medium volume of 0.20 ml/cm2. The maximum replicative life span of chick cells was obtained by serially propagating cells in a medium volume of 0.20 ml/cm2 containing 20 or 30% FBS, or, alternatively, in 0.53 ml/cm2 containing 10, 20 or 30% FBS. Cells grown in medium containing 5% serum had a calendar life span of 35 days, whereas cells propagated in medium containing higher serum concentrations had a calendar life span of 50 days. These results reenforce the concept that, although the kinetics of cell population aging can be affected by the culture medium composition, the aging of cells in culture is controlled by alterations within the cell.     

Ribosome biosynthesis in Tetrahymena pyriformis. Regulation in response to nutritional changes

Ribosome contents of growing and 12-h-starved Tetrahymena pyriformis (strain B) were compared. These studies indicate that (a) starved cells contain 74% of the ribosomes found in growing cells, (b) growing cells devote 20% of their protein synthetic activity to ribosomal protein production, and (c) less than 3% of the protein synthesized in starved cells is ribosomal protein. Ribosome metabolism was also studied in starved cells which had been refed. For the first 1.5 h after refeeding, there is no change in ribosome number per cell. Between 1.5 and 2 h, there is an abrupt increase in rate of ribosome accumulation but little change in rate of cell division. By 3.5 h, the number of ribosomes per cell has increased to that found in growing cells. At this time, the culture begins to grow exponentially at a normal rate. During the first 2 h after refeeding, cells devote 30-40% of their protein synthetic activity to ribosomal protein production. We estimate that the rate of ribosomal protein synthesis per cell increases at least 80-fold during the first 1-1.5 h after refeeding, reaching the level found in exponentially growing cells. This occurs before any detectable change in ribosome number per cell. The transit time for the incorporation of these newly synthesized proteins into ribosomes is from 1 to 2 h during early refeeding, whereas in exponentially growing cells it is less than 30 min. The relationship between ribosomal protein synthesis and ribosome accumulation is discussed.     

Effect of different fetal bovine serum concentrations on the replicative life span of cultured chick cells

Summary The effect of Eagle's minimal essential medium, containing different fetal bovine serum (FBS) concentrations, on the proliferation and replicative life span of cultured chick cells has been studied. Our results showed that the rate of chick cell proliferation and the cell density at stationary phase increased as a function of serum concentration between 5 and 30% FBS. The replicative life span of cultured chick cells was dependent on the FBS concentration between 5 and 20% in a medium volume of 0.20 ml/cm². The maximum replicative life span of chick cells was obtained by serially propagating cells in a medium volume of 0.20 ml/cm² containing 20 or 30% FBS, or, alternatively, in 0.53 ml/cm² containing 10, 20 or 30% FBS. Cells grown in medium containing 5% serum had a calendar life span of 35 days, whereas cells propagated in medium containing higher serum concentrations had a calendar life span of 50 days. These results reenforce the concept that, although the kinetics of cell population aging can be affected by the culture medium composition, the aging of cells in culture is controlled by alterations within the cell. This work was supported by IIT Research Institute.