Effect of fetal bovine serum on 3-methylcholanthrene-induced transformation of hamster cells in vitro

Summary Eighteen lots of fetal bovine serum were tested for their ability to support clonal growth and 3-methylcholanthrene-induced morphological transformation of hamster embryo cells in vitro. Most of them supported cloning efficiencies of over 11%. However, cloning efficiency alone was an inadequate criterion for selecting serum for transformation studies, since no transformation was observed with some lots, even though their cloning efficiencies were over 16%. This shows the importance of pretesting serum for its ability to support morphological transformation before it is used in mammalian cell carcinogenesis tests. Research sponsored by the National Cancer Institute under Contract No. N01-CO-75380 with Litton Bionetics, Inc.     

Inhibition of plasma membrane NADH oxidase activity and growth of HeLa cells by natural and synthetic retinoids

Several retinoids, both natural and synthetic, were evaluated for their ability to modulate NADH oxidase activity of plasma membranes of cultured HeLa cells and the growth of HeLa cells in culture. Both NADH oxidase activity and the growth of cells were inhibited by the naturally-occurring retinoids all trans-retinoic acid (tretinoin) and retinol as well as by the synthetic retinoids, trans-acitretin, 13-cis-acitretin, etretinate and arotonoid ethylester (Ro 13-6298). For all retinoids tested, inhibition of NADH oxidase activity and inhibition of growth were correlated closely. With tretinoin, etretinate and arotonoid ethylester, NADH oxidase activity and cell growth were inhibited in parallel in proportion to the logarithm of retinoid concentration over the range of concentrations 10-8 to 10-5 M. Approximately 70% inhibition of both NADH oxidase activity and growth was reached at 10 µM. With retinol, trans-acitretin and 13-cis-acitretin, inhibition of NADH oxidase activity and growth also were correlated but maximum inhibition of both was about 40% at 10 µM. The possibility is suggested that inhibition of the plasma membrane NADH oxidase activity by retinoids may be related to their mechanism of inhibition of growth of HeLa cells in culture. (Mol Cell Biochem 166: 101-109, 1997)     

Human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells

The potential of mesenchymal stem cells (MSC) to differentiate into different cell types has opened up the possibility of using these cells clinically to treat a variety of disorders. In this study we describe the use of human umbilical cord blood serum (CBS) as a replacement for fetal bovine serum (FBS) for culturing MSC from different sources. MSC from human and swine bone marrow and human umbilical cord blood were cultured in the presence of DMEM/F12 containing either FBS or CBS. Human MSC cultured in presence of FBS or CBS showed typical fibroblast-like morphology, which is characteristic of MSC. 99% of the cells cultured in FBS had a CD73+/CD105+/CD45- phenotype compared to 96% of cells cultured in CBS. Cells cultured in CBS had a significantly higher cell count as compared to cells cultured in FBS. Swine Bone Marrow MSC cultured in the presence of FBS and CBS were morphologically and phenotypically similar. Human umbilical cord blood serum supports the growth of MSC. While no significant differences were observed in the MSC numbers in swine cells cultured in the presence of FBS or CBS, human cells showed a greater proliferation potential in the presence of CBS as compared to FBS. Therefore, CBS can be used as an effective substitute to FBS for developing clinically useful protocols for culturing MSC.     

Effects of fetal bovine serum and serum-free conditions on white and dark axolotl neural crest explants

Summary Neural crest cells from both white mutant and dark (wildtype) axolotls (Ambystoma mexicanum) were cultured in increasing concentrations of fetal bovine serum (FBS; 2 to 20%). For each explant, the total number of cells that migrated and the percent of differentiated melanophores were recorded. At concentrations of FBS above 2% melanophore differentiation was essentially equivalent (32 to 59%) for both the white and dark neural crest cultures, but subtle differences in cell behavior and differentiation were found between the two phenotypes. By contrast there was a significant difference in the percent melanization of cells in serum-free control cultures, wherein melanophore differentiation in dark neural crest cultures was, on average, 18% compared to 5% in white cultures. Thus, contrary to all previously published work, white and dark neural crest cells are not intrinsically equivalent. Our culture results are discussed with regard to the probable in vivo conditions that cause the white phenotype. This research was supported by grant AR 34478 from the National Institutes of Health, Bethesda, MD, and a University of Kansas Biomedical Science support grant.     

Detection of squalene in alpha-fetoprotein and fetal serum albumin from bovine

Alpha-fetoprotein and fetal serum albumin have been simultaneously purified from fetal bovine serum by mild procedures utilizing ammonium sulfate, hydrophobic interaction, immobilized metal (nickel) affinity chromatography, and isoelectric focusing. The lipidic extract from each protein was analyzed by gas chromatography and the peak appearing just after the arachidonic acid was identified as squalene by gas chromatography-mass spectrometry. This isoprenoid was not detected formerly in these proteins from human, rat, bovine, and pig. Until recently, in the analysis of the fatty acid composition of the alpha-fetoprotein and serum albumin from mammals, a peak has been assigned in the last part of the chromatographic profile, after arachidonic acid, to docosahexaenoic acid. In the present work, it was found that the peak corresponds to squalene instead of docosahexaenoic acid. Furthermore, we conclude that bovine alpha-fetoprotein and fetal serum albumin carry squalene, but not docosahexaenoic acid. These results agree with others obtained analyzing the same proteins from chick embryo.     

Effects of defined medium,fetal bovine serum,and human serum on growth and chemosensitivities of human breast cancer cells in primary culture: Inference for in vitro assays

Summary We compared the effects of defined medium, fetal bovine serum (FBS) and human serum (HuS) on the growth and responses to chemotherapeutic agents of human breast cancer cells in primary culture. Normal and tumor tissues were dissociated to small aggregates and single cells and seeded onto collagen-gel-coated wells in defined medium or medium supplemented with 5% FBS or 5% HuS. In all cases examined, defined medium and medium containing HuS were superior to medium containing FBS in supporting growth of both normal and tumor cell cultures. However, cultures in defined medium showed an initial cell loss. Cells from the same tumor cultured in different media varied in their responses to chemotherapeutic agents. In light of these results, medium supplemented with HuS, which promoted attachment of these cells in culture and stimulated their growth, should be the most appropriate nutrient environment for determining the effects of therapeutic agents on cells as it most closely resembles the in vivo situation. Because there were also variations in growth rates and chemosensitivities of tumor cells cultured in different human serum samples, we suggest that optimal conditions in which to culture these cells include the serum of the patient whose tumor is removed. This serum may provide host factors that influence cell growth and interact with exogenous factors. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia. J. T. Emerman is a research scholar of the National Cancer Institute of Canada.     

The culture of human skin fibroblasts on microcarriers in the presence of serum substitute: Ultroser G

The effects of the substitution of serum by Ultroser G on human skin fibroblasts cultured on microcarriers were analysed. Cultures could not be established on microcarriers in the presence of Ultroser G. However, microcarrier cultures started in the presence of 10% foetal calf serum, and transferred to 2% Ultroser G after 7 days resulted in high cell densities.     

A unique property of fetal bovine serum: High levels of protein-glutathione mixed disulfides

Summary Fetal bovine serum has been reported to delay or inhibit “spontaneous” neoplastic transformation in vitro as compared with all other sera tested. The present results indicate that fetal bovine serum is also unique in containing high levels of protein-glutathione mixed disulfides (3 to 7 μg glutathione as mixed disulfide per ml serum). The level of mixed disulfide appears to vary in accordance with the period of gestation of the fetal calves used to prepare the serum, decreasing below detectable levels (less than 0.2 μg per ml) with nearterm fetal calves. Calf, adult bovine, fetal horse, and swine sera did not contain detectable levels of this type of mixed disulfide. This work was supported by a grant from the National Institutes of Health (CA 08348).     

Use of commercial serum replacements for the culture of insect cells

Summary Two commercially available serum replacements developed for use in the culture of hybridoma and other mammalian cells were tested for their suitability as replacements for fetal bovine serum in insect cell culture medium. CPSR-1 and CPSR-3 both supported growth of the insect cell line IPLB-SF-21AE. CPSR-3 supported adequate growth, but cells in medium supplemented with CPSR-1 grew much slower and achieved only about half the final cell density of either FBS or CPSR-3 supplemented medium. This work was supported in part by grant 187159 from the Juvenile Diabetes Foundation and BRSG RR05876 from the National Institutes of Health, Bethesda, MD.     

Fluorescence lifetime microscopy of the sodium indicator sodium-binding benzofuran isophthalate in HeLa cells

The behavior of the sodium indicator sodium-binding benzofuran isophthalate (SBFI) is investigated in HeLa cells by time-resolved fluorescence microscopy. The fluorescence relaxation of SBFI in HeLa cells can be described by a triexponential for intracellular sodium concentration ([Na(+)](i)) between 0 and 90 mM. Changes in [Na(+)](i) affect neither the fluorescence relaxation times (0.21, 0. 60, and 2.7 ns) nor the average decay time (2.2 ns). The preexponential factor of the shortest decay time is negative. However, the ratio of the fluorescence excitation signal at 340 nm to that at 380 nm increases with [Na(+)](i). To elucidate the behavior of SBFI in cells, experiments are performed on SBFI in buffer at various concentrations of sodium, potassium, and bovine serum albumin (BSA) and at various viscosities. The fluorescence decay is triexponential only in the presence of BSA. The relaxation times are independent of [Na(+)] and [BSA]. The preexponential factor of the shortest decay time is negative from a certain [BSA] on, which depends on [Na(+)]. The data indicate that interactions with intracellular components rather than microviscosity influence the SBFI behavior in cells. A model is suggested in which the fluorescence intensities are mainly determined by the signals from the Na(+) subsetSBFI and SBFI subsetprotein complexes.     

The effect of fetal bovine serum on polyene macrolide antibiotic cytotoxicity and antifungal activity

Summary The relationships between fetal bovine serum (FBS) concentration and polyene macrolide antibiotic cytotoxicity to animal cells and to fungi were evaluated. The toxicity of amphotericin B (AB) and its derivative, amphotericin B methyl ester (AME), toward KB cells was found to be directly related to fetal bovine serum concentration. At higher FBS levels, increased concentrations of AB and AME were required to reduce 72-hr KB viable cell numbers to 50% of control values. Similarly, polyene macrolide antibiotic levels required to inhibit the growth ofSaccharomyces cerevisiae to 50% of controls, and for obtaining minimum fungicidal concentrations (MFC), were greater when higher levels of FBS were used. In addition, AME was less toxic than AB toward KB cells grown in media containing 2, 5, 10, 15 or 20% FBS, whereas the antifungal activities of AB and AME were similar. AME was also capable of eliminatingCandida albicans, Saccharomyces cerevisiae, Aspergillus niger orFusarium moniliforme from KB cultures at antibiotic levels which exhibited less cell toxicity than did the concentrations of AB required for a similar response. These findings indicate that AME may be a potentially useful antifungal antibiotic for tissue culture systems. Portions of this paper were presented at the 25th Annual Meeting of the Tissue Culture Association at Miami, Florida, 1974. This investigation was supported in part by contract NIH 69-2161, NIH grant no. AI-02095 and NIH training grant no. GM 507 from the National Institute of General Medical Sciences.     

Glycoprotein and protein precursors to plasma membranes in vesicular stomatitis virus infected HeLa cells

Vesicular stomatitis virus is known to mature at HeLa cell plasma membranes. To study the process, cells, infected with vesicular stomatitis virus, were fractionated after short term labeling studies (1 min pulse, 1 min chase) to determine the assembly kinetics of G protein and M protein into plasma membranes. Newly synthesized M protein was found released in the supernatant from which free polysomes were sedimented during sucrose gradient analysis of these polysomes. If this M protein is particle bound, it must have a density of less than 1.08 g/ml. About 40% of this M protein so labeled was not sedimentable at 165,000 X g for 16 h. This newly synthesized M protein had not yet assembled into plasma membrane and thus must represent an internal pool. This and previous studies show that it has a subsequent transit time to the plasma membrane of about 2 min. Once associated with plasma membranes, M protein decayed in an approximately logarithmic fashion indicating that newly synthesized M randomly mixes (and turns over) with preexisting M protein. G protein was particle bound in a 1 min pulse, 1 min chase, and was never found released in a soluble form. At the later time when fucose is added to G protein, the oligosaccharide moiety is near to complete, and on completion is about 2,000 in molecular weight. Evidence is presented showing that fucose is probably attached to the N-acetylglucosamine of the protein carbohydrate linkage. G protein to which fucose had just been added was located internally on a membranous fraction of density 1.14 g/ml in sucrose; its subsequent transit time from this pool (which in uninfected cells is between 1–2% of the total cell fucosyl glycoprotein) was about 15 min. Because their densities were different and their transit times were different, internal newly synthesized M and fucosyl G protein which assemble into plasma membranes were not on the same internal membranous component. Association of M protein with the plasma membranes may thus occur from a nonsedimentable soluble cytoplasmic pool by a process of direct adsorption.     

Calcium oxalate crystals in fetal bovine serum: implications for cell culture, phagocytosis and biomineralization studies in vitro

Cell culture methods and models are key investigative tools for cell and molecular biology studies. Fetal bovine serum (FBS) is commonly used as an additive during cell culture since its constituents promote cell survival, proliferation and differentiation. Here we report that commercially available FBS from different major suppliers consistently contain precipitated, calcium oxalate crystals-either in the monohydrate (COM) or dihydrate (COD) form. Mineral structure and phase identification of the crystals were determined by X-ray diffraction, chemical composition by energy-dispersive X-ray microanalysis, and imaging and measurement of crystal growth steps by atomic force microscopy-all identified and confirmed crystallographic parameters for COM and COD. Proteins binding to the crystals were identified by immunoblotting, revealing the presence of osteopontin and fetuin-A (alpha(2)HS-glycoprotein)--known regulators of crystal growth found in serum. Macrophage cell cultures exposed to calcium oxalate crystals showed internalization of the crystals by phagocytosis in a process that induced disruption of cell-cell adhesion, release of reactive oxygen species and membrane damage, events that may be linked to the release of inflammatory cytokines by these cells into the culture media. In conclusion, calcium oxalate crystals found in commercially available FBS are toxic to cells, and their presence may confound results from in vitro studies where, amongst others, phagocytosis, biomineralization, renal cell and molecular biology, and drug and biomaterial testing are being examined.     

Effects of bovine colostrum, foal serum immunoglobulin concentration and intravenous plasma transfusion on chemiluminescence response of foal neutrophils

Summary. The effects of bovine colostrum, absorption of equine colostral immunoglobulins and age on phagocytic and serum opsonizing activity of nine clinically healthy foals were examined. Cells and serum were collected prior to suckling and at 7, 14 and 28 days of age. Seven foals had serum IgG concentrations >600mg/dl whereas two foals had <350mg of IgG/dl. Phagocytic and serum opsonic activity of eight clinically ill foals with <400mg of IgG/dl of serum were also examined before and after plasma transfusion. Phagocytic and serum opsonizing activities were evaluated by an assay for chemiluminescence (CL) after addition of opsonized streptococci. Results showed that bovine colostrum stimulated CL of foal neutrophils. Preliminary characterization of opsonins in bovine colostrum by ammonium sulphate fractionating and heat inactivation indicated that opsonins generating CL were mainly associated with immunoglobulin G. Chemiluminescence generated by foal neutrophils varied with age with foal neutrophils collected at day 14 producing more CL than adult neutrophils ( P <0.05). Foal serum opsonizing activity was similar to adult opsonizing activity if serum IgG concentrations were >600mg/dl but it was less if IgG concentration was <350mg/dl ( P <0.05). Chemiluminescence generated by foal and adult neutrophils was higher when post-transfusion foal serum was used as the source of opsonin than when pre-transfusion foal serum was used ( P <0.05). When adult serum was the opsonin, chemiluminescence of foal neutrophils collected before and after plasma transfusion did not differ. The increase in CL following plasma transfusion was probably due to an increase in serum opsonizing activity.     

Culture of an epithelial cell line (MDCK) with a serum substitute

This paper reports the suitability of culturing a line of dog kidney epithelial cells, MDCK, in the presence of a serum substitute, Ultroser G. Serial subcultivation with this product was possible for at least 10 passages without any change in cell shape and size, saturation density, dome-forming ability, transepithelial resistance, and growth curve. Adhesion of newly plated cells to plastic was somewhat lower than in fetal calf serum but the trypsin-harvesting kinetics were essentially the same. However, the membrane ion transport systems was alterd: cell sodium influx was greatly diminished, suggesting a deep change in the amiloride-sensitive Na⁺ channels: sodium efflux was highly enhanced (both active and passive).     

Suppression of the degradation of recombinant human apolipoprotein E by a protease inhibitor obtained from fetal bovine serum in serum-free culture

The recombinant human apolipoprotein E (Apo-E) produced by Chinese hamster ovary cells (CHO-322 cells) in serum free culture was degraded to 24K and 23K fragments that contained N-terminal amino acid. The degradation site of Apo-E to 24K fragment was between Arg180 and Leu181 and the C-terminal amino acid of 23K fragment was Gly169. In fetal bovine serum (FBS)-containing culture, the degradation was inhibited. However, in calf serum (CS) the inhibitory activity was not detected. Thus, we attempted the purification of the factor with this inhibitory activity from FBS. A protease inhibitor was purified to give a single peak from FBS by ammonium sulfate precipitation and combination of several column chromatographies. When this FBS-derived protease inhibitor (FBS-d-PI) was added to serum-free culture of CHO-322 cells, degradation of recombinant Apo-E to the 24K and 23K fragments was dose-dependently suppressed and accumulation of intact Apo-E in culture supernatant was observed. FBS-d-PI was found to be a glycoprotein with relative molecular size of 75K daltons under reducing condition, and 85K daltons under nonreducing condition by SDS-PAGE. A complex of FBS-d-PI and a cellular protease was also detected in culture supernatant by western blot analysis using mouse monoclonal antibodies against FBS-d-PI.     

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.     

Effects of bovine platelet-poor plasma on the proliferation of normal and transformed BALB/c 3T3 cells

Summary Platelet-poor plasma, as well as autologous platelet-rich serum, was prepared from freshly-drawn bovine whole blood. Bovine platelet-poor plasma had properties similar to those previously decribed for human platelet-poor plasma; e. g., it would (a) support the growth of virally transformed but not normal BALB/ c 3T3 cells, (b) act synergistically with either partially purified platelet-derived growth factor or fibroblast growth factor to initiate cell replication in quiescent 3T3 cells, and (c) act sequentially with platelet-derived growth factor to initiate 3T3 replication. It appears that bovine serum contains both competence and progression factors and that stimulation of fibroblasts with bovine serum involves at least two sequential stages analogous to those described for stimulation with human serum.     

Effect of steroid hormones on membrane profiles of HeLa S3 cells

The cervical carcinoma cell line, HeLa S3, was exposed to oestradiol and progesterone separately and combined in ratios similar to those of the luteal and follicular phases of the menstrual cycle. After 48 h of exposure, membrane proteins were extracted from the plasma membrane and analyzed. The lipid composition of the cells and lectin binding patterns to the cells were also assessed. The hormones had a profound effect on the HeLa S3 membrane protein profiles. Progesterone inhibited the synthesis of a wide variety of proteins while oestradiol negated this effect. Less variation between lipid profiles was noted although progesterone reduced the amount of cholesterol present. Differences in lectin binding profiles between the various treatments were noted, indicating an hormonal effect on glycosylation. Such hormonal effects on membrane composition may have implications in cervical susceptible to microbial infections.