Modulation of albumin-like protein and lysozyme production by bovine tracheal gland serous cells. Dependence on culture conditions

Bovine tracheal submucosal gland serous cells were cultured in medium supplemented with either 10% fetal calf serum or 2% Ultroser G, a commercial serum substitute for cell culture. The proteins synthesized and secreted into the culture medium during [35S]methionine pulse, chase and isoproterenol-stimulated periods were analyzed. Marked differences in the patterns of secretory radiolabeled proteins with Mr values ranging from 15,000 to 95,000 were observed between pulse and chase media of cells cultured in fetal calf serum and Ultroser G. In the presence of Ultroser G, albumin-like protein production was inhibited 95% as compared to cultures incubated with fetal calf serum. A bovine lysozyme-type enzymatic activity was detected only in medium from stimulated cells cultured in Ultroser G. The results suggest that bovine tracheal serous cells synthesize different proteins according to the composition of culture medium and release certain proteins when adrenergically stimulated.     

Long-term culture of fetal rat hepatocytes in media supplemented with fetal calf-serum Ultroser SF or Ultroser G

Fetal hepatocytes cultured in medium supplemented with fetal calf serum (FCS) or Ultroser SF do not maintain production of albumin or transferrin beyond one week of culture. When dexamethasone (10(-7) M) is present, secretion of albumin and transferrin can be extended to two weeks, however, levels are extremely low. By three weeks, neither plasma protein can be detected in the culture medium in either conditions of culture. In contrast, hepatocytes maintained in medium supplemented with Ultroser G continue to produce albumin and transferrin at high levels for the entire three week period of this study. The morphology of the cultures are different. In FCS and Ultroser SF supplemented medium there are many more fibroblast and epithelial-like cells and relatively fewer cells which are distinctly hepatocytes when compared with Ultroser G supplemented medium. The level of tyrosine aminotransferase, which is a dexamethasone inducible enzyme, is found to be much higher in Ultroser G cultures, with no further increase demonstrable by addition of dexamethasone. In contrast, dexamethasone induces the enzyme by about eight-fold in cultures maintained in FCS supplemented medium. Therefore it appears that Ultroser G already contains sufficient steroid activity to maximize the level of tyrosine aminotransferase. A comparison between Ultroser C and SF (steroid-free) suggests that the mixture of steroid and steroid derivatives in the G formulation must be important in the maintenance of differentiated functions of hepatocytes in culture. However, supplementation of FCS cultures with dexamethasone, which is known to be present in Ultroser G, does not allow hepatocytes to retain their differentiated functions over an extended period. Therefore it is concluded that other components besides dexamethasone must be important.     

Modulations of the epithelial phenotype and functional activity of cultured bovine tracheal gland cells: dependence on the culture medium and passage number.

Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse embryo fibroblast proliferation and prostaglandin production in medium supplemented with fetal bovine serum or serum substitutes (Ultroser SF and G): role of glucocorticoids

The growth of DBA/2 mouse embryo fibroblasts, as well as their prostaglandin (PG) production, was compared under 3 different culture conditions: RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2% Ultroser SF (steroid-free) or with 2% Ultroser G (containing steroids). The effect of the absence or presence of glucocorticoids on both parameters was more precisely investigated. In FBS-supplemented cultures, dexamethasone had a stimulatory effect on cells characterized by a slow growth rate, whereas it markedly inhibited proliferation in rapidly growing fibroblasts. The experiments carried out with serum substitutes (Ultroser SF and G) strongly corroborated the role of the absence or presence of glucocorticoids on fibroblast proliferation. Manipulations of glucocorticoid concentrations in Ultroser SF by adding 5 x 10(-8) M dexamethasone or in Ultroser G by adding 10(-6) M RU 486 reversed the effect of the absence of glucocorticoid in the first case, or in the latter case the effect of the presence of glucocorticoid on both cell growth and PG production. Progesterone had no effect by itself. Our results emphasize the importance of performing complete kinetic studies to investigate the effect of a given factor on cell proliferation in vitro, since glucocorticoids may have opposite effects on fibroblast proliferation according to their cell growth pattern in vitro.     

Neutral Red Assay for Measurement of Quantitative Vero Cell Cytotoxicity

A neutral red assay involving Vero cells was used to quantitate the cytotoxic activity of verotoxins (VT) produced by Escherichia coli and to investigate changes in titer caused by altering the composition of the cell culture medium. Three variations on medium 199 were investigated: one involved supplementing the medium with 5% fetal bovine serum (FBS), a second was the use of serum-free (SF) medium that contained 5% bovine serum albumin and 5 μg of fibronectin per ml, and the third involved the use of 4% Ultroser G, a commercial serum replacement. The level of cytotoxicity varied markedly with the type of VT and with the medium that was used. For VT1, there was no difference in cytotoxicity between medium with 5% FBS and SF medium, but cytotoxicity was reduced more than 100-fold in medium containing Ultroser G compared with cytotoxicity in the other media. For VT2, VT2v, and VTe, there was a slight reduction in cytotoxicity in medium containing 4% Ultroser G and a more marked reduction in SF medium compared with cytotoxicity in medium containing 5% FBS.     

Use of two replacements of serum during bovine embryo culture in vitro

This study evaluated the effect of two commercial serum replacements (Ultroser G and CPSR-3 on in vitro bovine embryo culture. In Experiment 1, zygotes were cultured in SOF+Ultroser G (2, 4 and 6%), SOF+CPSR-3 (2, 4 and 6%), and SOF+5% FCS (control). Blastocyst rates obtained after culturing with Ultroser G were lower than those with FCS. However, blastocyst rates for CPSR-3 were similar to those for serum. In addition, embryos produced in SOF+CPSR-3 had the same proportion inner cell mass number and total cell number as embryos cultured with FCS. In Experiment 2, a combination of serum replacements during different periods showed that treatment before the five-to eight-cell stages had no effect on further embryo development. However, treatments up to the morula stage affected blastocyst formation. The concentration of supplement and the timing of its inclusion in culture markedly affected embryo development. The serum replacement CPSR-3 can supplement embryo culture with blastocyst rates and quality similar to those for serum.     

Human vascular smooth muscle cells in culture: growth characteristics and protein pattern by use of serum-free media supplements

This study demonstrates that cultivation of vascular smooth muscle cells from human artery wall is possible under completely serum-free conditions. The effects of attachment factors on cell spreading and cell proliferation are described in detail as well as routine cultivation methods under serum-free conditions (clone cultures, cell migration, subcultivation by use of an exogenous trypsin inhibitor, cryopreservation and readaptation of cells). After a careful adaptation period, only two (BMS and Ultroser G) of the four commercially available serum-free media supplements tested were used successfully for a routine cultivation of the smooth muscle cells over several passages. With both supplements cell proliferation rates were comparable with those obtained in medium containing 10% fetal calf serum. The addition of platelet-derived growth factor or transferrin to serum-free cultures had no growth-stimulating effect. The addition of endothelial cell growth factor isolated from bovine brain caused a significant increase in proliferative activity of cells cultivated with BMS, but not with Ultroser G. Moreover, we report that under the serum-free culture conditions described here, the gamma-actin content of the cells is largely reduced (51% +/- 13% (means +/- SD) for cells cultivated in Ultroser G, and 12% +/- 4% (means +/- SD) for cells cultivated in BMS) when compared with cells cultivated under serum-containing conditions (gamma-actin content = 100%). The alpha-actin content was observed to be unaltered. Even after a careful readaptation of serum-free cultured cells to serum conditions, the gamma-actin content remained reduced.     

Culture of chondrocytes in medium supplemented with fetal calf serum or a serum substitute: Ultroser G

Fetal calf serum and a serum substitute, Ultroser G, were compared for their effects on the growth curves, clonal growth and cell cycle progression of rabbit chondrocytes in primary culture and during at least three cell passages and included a screen for the maintenance of cartilage-like differentiation i.e. the presence of type II collagen. Proliferation was also compared with another serum substitute, Nu-Serum. Ultroser G is shown to be equivalent to fetal calf serum as far as chondrocyte proliferation is concerned, clonal growth is improved and biosynthesis of type II collagen is maintained in primary culture.     

Cultivation of HeLa cells with fetal bovine serum or ultroser G: Effects on the plasma membrane constitution

Summary Plasma membranes isolated from HeLa cells cultivated in suspension cultures supplemented with 3.5% fetal bovine serum or 2% of the commercially available serum substitute Ultroser G contained the same amounts of protein, cholesterol, and phosphate on a cellular basis. Minor differences in the plasma membrane fatty acid composition were seen, with the most pronounced alteration observed for palmitic acid, which amounted to 27 and 20% in fetal bovine serum- and Ultroser G-supplemented cells, respectively. Plasma membranes from cells growth with Ultroser G contained almost twice as much phosphatidylethanolamine and displayed two thirds of the phosphatidylcholine content, compared to plasma membranes obtained from fetal bovine serum supplemented cells. The former membranes also showed a 3 times higher specific [³H]acetate labeling of cholesterol, indicating a higherde novo synthesis of cholesterol. Both quantitative and qualitative alterations were revealed among the plasma membrane polypeptides when these were subjected to immuno- and lectin blottings. Fluorescence anisotropy measurements at different temperatures produced similar results irrespective of the growth medium supplement when the plasma membrane specific probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene was used on intact cells. However, the average cellular rigidity was higher for Ultroser G supplemented cells, determined with 1,6-diphenyl-1,3,5-hexatriene as a probe. This investigation was supported by grants from the Swedish Natural Science Research Council, Anders Otto Sw?rds Stiftelse, Stockholm, Crafoordska Stiftelsen, Lund and Kungl. Fysiografiska S?llskapet, Lund.     

Serial subcultivation of Giardia lamblia in Keister's modified TYI-S-33 medium containing ultroser G.

The ability of Giardia strains of the duodenalis type to grow in Keister's modified TYI-S-33 medium varies with serum lot. Recently, strains of Giardia including MR4, WB, and Human-1-Portland, have been cultivated in Keister's modified TYI-S-33 medium containing the serum substitute Ultroser G and have been cultured serially as least 40 times. An optimal concentration of 8% Ultroser G promotes maximal growth in Keister's modified TYI-S-33 medium for all three strains. This concentration of Ultroser G will produce a two-log increase in the number of trophozoites in approximately three days post-inoculation. Generation times for the trophozoites ranging from 6 to 11 h have been achieved in Keister's modified TYI-S-33 containing 10% adult bovine serum and from 8 to 13 h in Keister's modified TYI-S-33 with 8% Ultroser G. Despite the excellent growth of Giardia strains in medium containing Ultroser G, the maximum trophozoite density is only about half of that achieved in Keister's modified TYI-S-33 medium supplemented with 10% adult bovine serum. Comparisons of trophozoites grown with serum or the serum substitute reveal no discernable differences in morphology and motility. Additionally, these strains have been successfully cryopreserved and revived in Keister's modified TYI-S-33 medium supplemented with Ultroser G. Because Ultroser G is a characterized mixture of six main groups of ingredients (growth factors, adhesion factors, mineral trace elements, hormones, binding proteins, and vitamins), the variability in cell proliferation that may occur when changing serum lots should be minimized when using this product.     

Differentiation markers of mouse C2C12 and rat L6 myogenic cell lines and the effect of the differentiation medium

Summary The differentiation grade of cells in culture is dependent on the composition of the culture medium. Two commonly used myogenic cell lines, mouse C₂C₁₂ and rat L₆, usually differentiate at a low concentration of horse serum. In this study we compared the effect of horse serum with a medium containing a low percentage of Ultroser G and rat brain extract. The maturation grade was evaluated on the basis of various biochemical, (immuno)histochemical and cell-physiological parameters. Substitution of horse serum by Ultroser G and rat brain extract during the differentiation phase resulted in a higher maturation grade of the myotubes of both cell lines, on the basis of creatine kinase activity and the diameter of the myotubes. In addition, the C₂C₁₂ myotubes display cross-striation, contain a higher percentage of creatine kinase muscle-specific isoenzyme MM, show a ninefold increase in acetylcholine receptor (AChR) clusters, form a continuous basement membrane, and have a lower resting cytosolic Ca²⁺ concentration. L₆ myotubes show a fivefold increase in AChR clusters and a twofold increase in the expression of the mRNA of the ɛ-subunit of AChR. C₂C₁₂ cells show spontaneous contraction and response of cytosolic Ca²⁺ to various stimulants in contrast to L₆ cells which do not. These studies established that the Ultroser G/brain extract medium leads to a higher differentiation grade of both cell lines, but parameters appropriate for use as differentiation markers appear to differ between both cell lines.     

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.     

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.     

Serial Subcultivation of Giardia lamblia in Keister's Modified TYI-S-33 Medium Containing Ultroser G

ABSTRACT. The ability of Giardia strains of the duodenalis type to grow in Keister's modified TYI-S-33 medium varies with serum lot. Recently, strains of Giardia including MR4, WB, and Human-1-Portland, have been cultivated in Keister's modified TYI-S-33 medium containing the serum substitute Ultroser G and have been cultured serially at least 40 times. An optimal concentration of 8% Ultroser G promotes maximal growth in Keister's modified TYI-S-33 medium for all three strains. This concentration of Ultroser G will produce a two-log increase in the number of trophozoites in approximately three days post-inoculation. Generation times for the trophozoites ranging from 6 to 11 h have been achieved in Keister's modified TYI-S-33 containing 10% adult bovine serum and from 8 to 13 h in Keister's modified TYI-S-33 with 8% Ultroser G. Despite the excellent growth of Giardia strains in medium containing Ultroser G, the maximum trophozoite density is only about half of that achieved in Keister's modified TYI-S-33 medium supplemented with 10% adult bovine serum. Comparisons of trophozoites grown with serum or the serum substitute reveal no discernable differences in morphology and motility. Additionally, these strains have been successfully cryopreserved and revived in Keister's modified TYI-S-33 medium supplemented with Ultroser G. Because Ultroser G is a characterized mixture of six main groups of ingredients (growth factors, adhesion factors, mineral trace elements, hormones, binding proteins, and vitamins), the variability in cell proliferation that may occur when changing serum lots should be minimized when using this product.     

Subculture of rabbit articular chondrocytes within a collagen gel: growth and analysis of differentiation

Primary cultures of rabbit articular chondrocytes have been subcultured within three-dimensional (3D) collagen gels. Under these conditions, the cells remained viable and divided, but with a lower proliferation rate than that observed in control monolayer cultures. Flow cytometric analysis of progression of the cells into the cell cycle has confirmed and extended these findings. Also the cellular volume was decreased in 3D-culture, being in the same range as thein vivo size of cartilage cells. Specific staining for proteoglycans and type II collagen immunolocalization on sections of gels showed the expression of differentiated phenotypes and revealed the accumulation of these matrix components in the immediate surroundings of the cells. The use of Ultroser G (a serum substitute) improved the conditions for 3D- culture of rabbit articular chondrocytes.     

Ultroser G and brain extract induce a continuous basement membrane with specific synaptic elements in aneurally cultured human skeletal muscle cells

Basement membrane (BM) components were studied on human muscle and skeletal muscle cells cultured on different media by immunofluorescence and electron microscopy. Their topographical relation with acetylcholine receptors was investigated. Myotubes cultured on a combination of the serum substitute Ultroser G and brain extract show a continuous layer of heparan sulfate proteoglycans (HSPGs), laminin, and type IV collagen. In contrast, myotubes cultured on serum-containing media are associated with granular depositions of HSPG and laminin and only with wisps of type IV collagen. Omission of brain extract or substitution by chicken embryo extract results in an intermediate staining pattern. For all types of cultures, fibronectin is localized in and around mononuclear cells, but hardly associated with myotubes. A codistribution between clusters of acetylcholine receptors and HSPG and laminin and Vicia villosa B4 lectin-positive material exists only in Ultroser G/brain extract-based myotubes like in muscle in vivo. No clustering is observed in serum-based myotubes. Electron microscopy reveals that the former myotubes are surrounded by a continuous BM consisting of a lamina lucida, lamina densa, and lamina fibroreticularis. Proteoglycans are present on the external site of the lamina densa and associated in a regular fashion with collagen fibrils. In conclusion, BMs associated with myotubes cultured on Ultroser G/brain extract resemble in many ways the in vivo situation, including synaptic specializations. Cultured myotubes may serve as a model system for studies on the structure and function of human muscular (synaptic) BM under normal and pathological conditions.     

The biochemical and structural maturation of human skeletal muscle cells in culture: The effect of the serum substitute Ultroser G

On the basis of the percentage creatine kinase-MM, human skeletal muscle cells cultured on growth and differentiation media containing the serum substitute Ultroser G reach a significantly higher maturation grade after 7 days of differentiation than cells cultured on serum-containing media. They also remain viable for longer periods. The myotubes are much longer, their nuclei are often localized in rows on the periphery, and they show cross-striation more frequently. The activities of creatine kinase, citrate synthase, cytochrome c oxidase, AMP deaminase, and phosphorylase are significantly higher. Extending the differentiation period to 3 weeks increases the maturation grade of the cultures and the activities of all the enzymes mentioned before, except phosphorylase. A correlation exists between the enzyme activities and the maturation grade of the muscle cells. The content of fatty acid-binding protein also increases significantly with the maturation grade in contrast to the palmitate oxidation rate. The AMP deaminase and creatine kinase activity and the percentage MM-type remain lower in cultured cells than in adult muscle and the hexokinase activity remains higher, but the other enzyme activities become comparable after 20 days of differentiation. The myotubes, derived from Ultroser G-containing culture media, show spontaneous contractions after 12 days and cross-striation after 20 days when immunostained for the M-subunit of creatine kinase. These cells possess clusters of acetylcholine receptors, but aggregation of desmin at the site of the clusters was never detectable. The possibility of cultivating muscle cells with a predictable maturation grade allows the study of muscle development and muscular diseases caused by differentiation defects or by deficiency of a maturation-dependent (iso)enzyme.     

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).     

Evaluation of serum‐free differentiation conditions for C2C12 myoblast cells assessed as to active tension generation capability

We have compared several serum‐free media for the differentiation of C2C12 myoblasts and assessed the extent of differentiation in several ways including as to active tension generation capability. C2C12 cells were allowed to differentiate in Dulbecco's modified Eagle's medium (DMEM) containing Ham's F‐12 (F‐12), AIM‐V (AIM), 0.2% Ultroser‐G in DMEM (Ult‐G), and 0.1% Sericin in DMEM (Sericin), compared with in DMEM supplemented with 2% horse serum (HS) or 2% calf serum (CS). C2C12 differentiation was assessed as the extent of myotube formation, glucose metabolism, protein expression, sarcomere formation, and active tension generation. All serum‐free media examined were capable of inducing myotube formation and the expression of muscle‐specific proteins. All serum‐free media except for F‐12 gave the sarcomere structure. Active tension generation was observed for cells that differentiated in AIM and Ult‐G, but the active tension generated by C2C12 cells that differentiated in Ult‐G was only ～25% in the case of myotubes that formed in HS. The addition of Ult‐G to the AIM resulted in improvement of the active tension generation capability, the active tension generated being ～3.4× compared to that in HS. The approach for assessing muscle cell differentiation presented in this study will be suitable for other studies that involve the differentiation of muscle cells. Biotechnol. Bioeng. 2010;107: 894–901. © 2010 Wiley Periodicals, Inc.     

Nuclear and chromosomal replication patterns in chorionic villi cells by bromodeoxyuridine labelling and DNA flow cytometry

Cell kinetics of chorionic villi (CV) were studied by BrdUrd-incorporation detected by fluorescence-plus-Giemsa and BrdUrd-antibody techniques, and by DNA flow cytometry. Growth characteristics of long-term cultures of CV resembled fibroblasts with a total cell cycle time of 26 h, final S phase of 9 h, penultimate S phase of 16 h and G2/M phase of 3-4 h. Especially useful for a quick routine diagnostic approach, Ultroser RG, a commercially available serum supplement, significantly increased cell proliferation and stabilized cell cycle lengths to a total cell cycle time of 14 h, final S phase of 7 h, penultimate S phase of 6 h and G2/M phase of 4 h. Moreover, mitotic activity steadily increased in cultured CV, when studying six successive subculturings. This reflects adaptation to the culture conditions rather than an inherent response of cultured CV cells of increasing passage numbers. Native villi exposed to BrdUrd immediately after biopsy show lower rates of uptake than do aliquots after overnight incubation. As shown by BrdUrd-pulse labelling studies, more than 7 h are required to overcome the proposed 'biopsy stress'. This correlates with routine diagnostic techniques, in which many more metaphase cells are observed in short-term cultures than in direct preparations.