Cumulative population doublings as the determinant of chick cell lifespan in vitro

Chick embryo fibroblasts were maintained at confluency for up to 35 days in medium containing 0.5% or 0.75% fetal bovine serum or 2.5% or 5.0% horse serum. At weekly intervals cells were subcultured and serially propagated in medium containing 10% FBS until their replicative lifespans were completed. The results showed that the replicative lifespan of embryonic chick fibroblasts was dependent on the cumulative number of population doublings undergone by the culture and was not related to the calendar time cells were in culture. Further characterization of 0.75% FBS maintained chick cells returned to 10% FBS medium showed that cells had protein contents and incorporated ³H-thymidine into DNA at a rate that resembled that of young cells, despite an advanced chronological age.     

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.     

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.     

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

Summary We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.     

A cobblestone cell isolated from the human omentum: the mesothelial cell; isolation,identification, and growth characteristics

Summary Normal human mesothelial cells (NHMC) were isolated from pieces of human omentum. The cell yield was approximately one million cells per square centimeter omentum. The mesothelial cells were identified by their positive staining with monoclonal antibodies against cytokeratins 6 and 18. Transmission electronmicroscopy of cultured NHMC revealed many microvilli on the apical surface and many mitochondria and pinocytotic vesicles in the cytoplasm, indicating active transmembrane transport. Growth of NHMC was directly related to the concentration of human serum or of fetal bovine serum in the growth medium. Addition of epidermal growth factor with or without hydrocortisone resulted in a significant increase of NHMC growth; when endothelial cell growth factor, insulin, or hydrocortisone were added no such increase was observed. Seeding NHMC at densities less than 3000/cm² did not result in monolayer formation. The mesothelial cells were serially passed in growth medium M199 with added 10% fetal bovine serum up to 7 passages. However, after Passage 4 the cells changed into giant cells with an irregular pattern, and a lack of intracellular cytokeratin expression was observed for most of the cells.     

Characterization of proliferation and differentiation of opossum kidney cells in a serum-free defined medium

Summary Proliferation and differentiation of opossum kidney cells in a serum-free defined medium was investigated and compared to that under conditions in which fetal bovine serum FBS (10%) was employed. Monolayers were grown in Dulbecco's modified Eagle's medium-Ham's F12 nutrient mixture containing insulin (10 μg/ml), bovine serum albumin fraction V (1 mg/ml) and fetuin (1 mg/ml). Cells in serum-free medium seeded at 1×10⁴ per cm² grew to confluency within 6 to 8 d and formed hemicysts or domes at a frequency equivalent to those in serum-containing medium. Electron microscopy of cultures grown in serum-free medium revealed polarized monolayers with the presence of microvilli and tight junctions. The differentiated characteristics, including sodium-dependent phosphate transport, the inhibition of this transport by parathyroid hormone (PTH), and the generation of cyclic AMP in response to PTH, were preserved in opossum kidney cells grown in serum-free medium.     

Senescence and cell density of human diploid fibroblasts influence metabolism of insulin-like growth factor binding proteins

In order to analyze changes in metabolism of insulin-like growth factor binding proteins (IGFBPs) related to cell senescence and cell density, we compared human diploid fibroblasts (HDF) in the proliferatively vigorous first half (young cells) and senescent HDF in the last 10% (old cells) of the replicative lifespan after seeding cells over an eighfold range and proliferation to high density. Increasing the seeding cell density of both young and old HDF led to elevated rates of IGFBP-3 secretion, an increasing ratio of the 42/38 kDa species of IGFBP-3, and degradation of all species of IGFBPs derived from both the fetal bovine serum component of the culture medium and from HDF. At a given seeding density old HDF produced more IGFBP-3 and degraded more IGFBPs than young HDF. IGFBP-4 was degraded by a protease that appeared to be different from the protease(s) involved in degradation of the other IGFBPs. Young HDF at all seeding densities contained a cell-associated 29 kDa IGFBP, whereas this protein could not be detected in old cells. Thus, although certain changes in IGFBP metabolism are similar in young HDF seeded at high densities and in old HDF, young and old phenotypes can be distinguished by characteristic qualitative and quantitative changes in IGFBPs derived from fetal bovine serum and from HDF. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

     

Proliferative characteristics of clonal endothelial cell strains

We have utilized clonal strains of bovine fetal aortic endothelial cells to study cellular senescence in a differentiated cell type of physiological significance. Serial subcultivation of nine endothelial clones derived from three fetal calf aortas revealed proliferative life-spans in vitro of 53–125 population doublings (PDs), compared with 60 and 143 PDs for two lines of bovine fetal lung cells and 85 and 147 PDs for two lines of bovine vascular smooth muscle cells. Serial growth curves showed marked reductions associated with endothelial cellular senescence both in cellular growth rate and culture plateau density. Studies of the 24-hour [³H]-thymidine labeling index versus percentage of proliferative life-span completed indicated that clonal endothelial cultures contained a large proportion (greater than 90%) of rapidly cycling cells until about 75% of the life-spans were completed. Senescent endothelial cells showed evidence of large increases in cell area, cell volume, and protein content. In those clones examined, one specialized endothelial function, Factor VIII antigen expression, was retained qualitatively throughout the life-spans.     

The establishment and characterization of mouse L-929 cells in protein-free Eagle's Minimal Essential Medium

The mouse cell line L-929 was established in protein-free Eagle's Minimal Essential Medium. The cells have been 'adapted' to continuous growth in the medium using stepwise reductions in the concentration of fetal bovine serum. The cells designated L-929-WS have now been propagated in protein-free Eagle's Minimal Essential Medium for two years. The population-doubling time was about 37 h. The addition of serum stimulated cell growth only slightly, but the saturation density was significantly increased. Morphological examination, a study of the secretion of colony stimulating activity and cytochemical investigations for acid phosphatase and alkaline phosphatase showed that L-929-WS cells, grown in protein-free Eagle's Minimal Essential Medium, did not differ markedly from cells propagated in medium containing serum. The cells provided a simple model for the study of cell growth in the absence of serum or the other macromolecular substances usually added to cell cultures. The general application of the cells for purposes in which the addition of serum or growth factors might interfere, is suggested.     

Establishment and characterization of a cell line derived from bovine tracheal glands

Summary Bovine tracheal submucosal gland cells have been isolated by enzymatic digestion and serially propagated in tissue culture for more than 12 mo. (40 passages). The cells exhibit an epithelioid appearance at confluence and contain alcian blue (pH 2.5)/periodic acid-Schiff-positive material within cytoplasmic granules. By electron microscopy numerous osmiophilic secretory granules are seen. Maximal growth is observed when the cells are grown on human placental collagen-coated culture vessels in medium supplemented with 20% fetal bovine serum. Scintillation spectrometry revealed that radiolabeled precursor (³⁵SO₄) was incorporated into high molecular weight molecules and released from cells. Isoproterenol (10⁻⁶ to 10⁻³ M) stimulated the release of³⁵SO₄. The maximal response to isoproterenol was completely inhibited by the β-adrenergic antagonist propranolol. It is concluded that the cultured cells retain features of tracheal gland cells and may serve as a useful model of synthesis and secretion of macromolecules by tracheal gland cells. This study was supported in part by NIH Program Project grant HL-24136, by a National Cystic Fibrosis Foundation Research Development Grant, and by a grant from Cystic Fibrosis Research, Inc. Dr. Finkbeiner is a recipient of NIH Clinical Investigator Award HL-01387.     

Changes in serum influence the fatty acid composition of established cell lines

Summary The fatty acid composition of different kinds of commercially available serum used to supplement cell culture media differs widely. As compared with fetal bovine serum, horse and bovine calf serum have a very high content of linoleic acid (18:2) and are low in arachidonic acid (20:4). (Fatty acids are abbreviated as number of carbon atoms: number of double bonds). Swine serum contains substantial amounts of both 18:2 and 20:4. Only fetal bovine serum contains more than 1% docosahexaenoic acid (22:6). Considerable differences in fatty acid composition occur when cells are grown in media containing any of these different serum supplements. The 18:2 and 20:4 content of 3T3 mouse fibroblast phospholipids is highest when the medium contains horse serum, intermediate with bovine calf serum, and lowest with swine or fetal bovine serum. Likewise, the highest phospholipid 18:2 content in Madin-Darby canine kidney cells (MDCK) occurs when the medium contains horse serum. With MDCK cells, however, growth in swine serum produces the highest 20:4 content. The 3T3 cell phospholipids accumulate more than 1% 22:6 only when the medium contains fetal bovine serum, whereas in no case do the MDCK cell phospholipids accumulate appreciable amounts of 22:6. The fact that the cellular fattyacid composition is likely to change should be taken into account when changes are contemplated in the serum used to grow established cell lines. These studies were supported by Arteriosclerosis Specialized Center of Research Grant HL 14,230 from the National Heart, Lung, and Blood Institute, National Institutes of Health.     

Culture of human blood vessel endothelial cells--a simple and inexpensive culture method

The purpose of this review is to introduce a simple and inexpensive culture method for human umbilical blood vessel endothelial cells. The medium used is MCDB-104 supplemented with 10% fetal bovine serum, 70 ng/ml endothelial cell growth factor from new-born bovine brains, 10 ng/ml murine epidermal growth factor, and 100 micrograms/ml heparin. The culture dishes are coated with gelatin. Under these conditions, endothelial cells from human vessels were grown with doubling times of 18-22 hrs and reached saturation densities of 8-12 x 10(4) cells/cm2. To determine the lifespan of the endothelial cells, the cells were serially subcultivated weekly at an inoculum size of 1,000 cells/cm2. Human endothelial cells from umbilical vein and artery were grown for 21 to 37 passages with 55 to 125 population doublings. This culture method seems to be useful for studying cell proliferation and functions of human endothelial cells.     

Ectopic production in serum-free media of the common alpha subunit of the glycoprotein hormones

Summary The HeLa-S₃ cell strain grown in Ham's F12 medium supplemented with insulin, transferrin, cortisol, epidermal growth factor, fibroblast growth factor, and trace elements, but containing no serum, continued to produce the common α-subunit of the glycoprotein hormones for the 10 d study. The amounts of α-subunit secreted into the medium during the first 4 d were indistinguishable from those in F12 medium supplemented with 10% fetal bovine serum. During the remainder of the experiment the amounts of α-subunit reached 50 to 80% those in the serum-supplemented medium.     

Failure of hydrocortisone or growth factors to influence the senescence of fibroblasts in a new culture system for assessing replicative lifespan

It has been reported that the replicative lifespan of human fibroblasts can be substantially extended by supplementing the growth medium with hydrocortisone or increased levels of serum proteins. These observations have been made only on cell populations transferred many times at high cell density, and cumulative population doublings have been recorded, rather than a more direct measure of cell division potential. We have measured the replicative potential of human fibroblasts cultured so as to avoid conditions of high cell density, medium depletion, and departure from exponential growth. Two fetal lung and two newborn foreskin fibroblast strains were serially passaged in the presence or absence of hydrocortisone (HC), epidermal growth factor (EGF), and fibroblast growth factor (FGF) until they senesced. At each passage cells were plated at densities sufficiently low that colony-forming efficiency could be calculated. We determined cumulative population doublings and also estimated the number of cell generations attained under each condition. FGF caused small but possibly significant changes, while HC and EGF failed to substantially alter replicative lifespan. The reported effect of HC on the doubling potential of fetal lung fibroblasts is therefore not an inevitable action of this hormone on the senescence mechanism, but may instead depend for its apparent activity on the passage regimen used. The fibroblast's insensitivity to EGF as a modulator of replicative potential, as compared with the keratinocyte, whose lifespan can be tripled by EGF, implies that the mechanisms limiting the replicative potential of these two cell types are not identical.     

Long-term culture of human endothelial cells

Summary Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10³/cm²) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts. This research was supported by grants from the U.S. Public Health Service (AG 01732, HL 16387, and HL 07080), the Cystic Fibrosis Foundation, and the New York and American Heart Associations. Victor B. Hatcher is an Established Fellow of the New York Heart Association and a recipient of the Ann Weinberg Cystic Fibrosis Research Scholarship Award.     

Clonal growth of normal human uroepithelial cells

Summary We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells (HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency (CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3×10⁵ lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum. Using these conditions, the average CFE was 16.1±2.5%. A cloning efficiency of 4.9±1.5% was obtained under the same conditions in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder cells resistant to 5 μg/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone supports HUC clonal growth with a CFE of 17.9±2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented MCDB 170 medium containing 70 μg/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7±1.7%. This work was supported by NIH grant 29525 to C. A. R. L. J. L. is a recipient of National Science Foundation predoctoral fellowship.     

Effects of growth conditions on cyclic nucleotide phosphodiesterases of cultured fibroblasts.

Cyclic nucleotide phosphodiesterase activities of baby hamster kidney cells (BHK) grown in surface cultures were altered by modifying growth conditions. The untransformed BHK cells grown in medium containing 10% fetal calf serum showed non-linear LineweaverBurk plots for cyclic AMP phosphodiesterase activity with apparent Michaelis constants for cyclic AMP of approximately 5 and 30 muM. When these cells were placed in medium containing 1% fetal calf serum, linear kinetic plots for cyclic AMP phosphodiesterase with an apparent Km for cyclic AMP of approximately 20 muM were obtained. Modification of the apparent Km of BHK cell phosphodiesterase was detectable within 20 minutes after dillution of cells grown in 10% serum into fresh medium containing 1% serum. With the BHK cell line transformed with Rous sarcoma virus, differences in enzyme kinetics were not seen when these cells were diluted in 1% or 10% serum. In addition to the serum induced differences in the apparent Km of cyclic AMP phosphodiesterases of BHK cells, total cyclic AMP and cyclic GMP phosphodiesterase activities were also modified by growth conditions. BHK cells grown to high cell densities had three to five-fold higher total cyclic AMP activity than did the cells in less dense cultures. When the dense cell cultures were diluted into fresh medium containing 10% serum, total enzyme activities fell to levels comparable to those found in the rapidly growing cells at low cell densities. The reduction in total enzyme activity after dilution of BHK cells occurred rapidly and was influenced by cell density. A similar reduction of total enzyme activity was also seen in diluted RSV cells; however, the time course of the response differed from that seen in the untransformed cells.     

The influence of cell population density and serum on the (Na+K)-ATPase of 3T3 cells

The (Na+K)-ATPase of 3T3 cells has been measured as a function of culture density and the type of serum in which the cells were grown. When 3T3 cells were grown in medium containing 10% newborn calf serum their (Na+K)-ATPase activity increased as the culture density increased and the cells became quiescent at densities of about 17.5 X 10(4) cells/cm2. When these cells are subcultured into medium containing 10% foetal bovine serum the activity of the enzyme decreases as the culture density increases and the cells attain final densities of about 5 X 10(4) cells/cm2.     

Trypanosoma cruzi: intracellular stages grown in a cell-free medium at 37 C.

A liquid medium containing a high concentration of water-soluble vitamins and ATP was developed for serial cultivation of Trypanosoma cruzi at 27–37 C; fetal bovine serum and trypticase were the only undefined substances in this medium. At 27 C, Trypanosoma cruzi grows primarily (over 99%) as epimastigotes with a population density reaching 92.7 × 10⁶/ml after 12 days of incubation. During the first subculture at 37 C, many epimastigotes from the original inocula changed into metacyclic trypomastigotes after 48 hr; the trypomastigotes subsequently transformed into amastigotes by 96 hr. In the second passage at 48 hr, 57.8% of the organisms were trypomastigotes which changed into amastigotes by the end of the incubation period. The proportion of amastigotes in the third and subsequent passages increased steadily as the proportion of epimastigotes gradually diminished. Amastigotes thus obtained could be serially subcultured indefinitely, yielding population densities of over 3.0 × 10⁷/ml of medium in 4–5 days at 37 C. Available evidence indicates that these amastigotes are morphologically and physiologically similar to intracellular amastigotes.     

Effect of fractions of horse and fetal bovine serum on neoplastic conversion of C3H mouse cells in tissue culture

Summary Cultures derived from C3H/He mouse embryos were grown in medium NCTC 135 supplemented with horse serum, fetal bovine serum, or various combinations of large and small molecule fractions of horse and fetal bovine serum. Cultures in medium NCTC 135 alone or in medium 135 supplemented with the small molecule fraction of either horse or fetal bovine serum did not grow as continuous long term lines. The best growth was obtained when the cultures were in medium containing the large molecule fraction of fetal bovine serum either alone or in combination with a small molecule fraction. Cells grown in the presence of the low molecular weight fraction of horse serum invariably produced tumors on injection into syngeneic animals. Cells in the small molecular weight fraction of fetal bovine serum combined with the large molecular weight fraction of horse serum produced tumors after a prolonged period in vitro. *** DIRECT SUPPORT *** A00S8010 00003