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)     

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.     

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.     

Growth and characterization of isolated bovine tracheal gland cells in culture. Influence of a reconstituted basement membrane matrix

We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth-Holm-Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37 degrees C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes. Enzymatic digestion using only trypsin, centrifugation and inactivation steps were repeated three times. Using this protocol, we obtained 15 +/- 4 (X 10(6] cells per g of tracheal submucosa with 72 +/- 2% (n = 5) cell viability. On microscopic observation, isolated cells were mainly composed of serous type glandular cells. Cells were cultured in a 1:1 medium of Dulbecco's Modified Eagle's/Ham's F12 supplemented with 10% fetal calf serum and subcultured in either plastic flasks or flasks coated with EHS matrix. On the plastic, the BTG cells exhibited at confluency an epithelioid appearance. They stained positively with the immunofluorescent anticytokeratin antibody and contained PAS-staining granules. By electron microscopy, lactoferrin, a protein marker specific to the serous cells, was demonstrated immunocytochemically in small secretory vesicles. BTG cells cultured on EHS matrix revealed a significantly increased growth in comparison to those cultured on plastic. In post-confluent culture of BTG cells on EHS matrix, we observed numerous dome-like structures formed by differentiated cells which were joined together around luminal spaces.     

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.     

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.     

The use of Ultroser G as a serum substitute in the culture of human skin fibroblasts

Growth of human skin fibroblasts was dramatically enhanced when serum in the culture medium was replaced by Ultroser G. Compared to the same cells cultured in the presence of serum, alterations in glucose and lipid metabolism and an increase in the activity of prolidase (EC 3.4.13.9) and prolinase (EC 3.4.13.8) were also observed. Consequently, we advise extreme caution in the use of Ultroser G in metabolic studies, especially for periods of culture exceeding 10 days. However, Ultroser G can help to produce a large number of cells and so facilitate purification of the proteins produced during the stationary growth phase.     

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.     

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.     

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.     

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.     

Culture of endocrine pancreatic cells in protein-free,chemically defined media

Summary Cell suspensions prepared by collagenase digestion of pancreata obtained from 21.5-d-old rat fetuses were preincubated in RPMI medium containing 10% fetal bovine serum (FBS), to ensure cell adhesion. Twenty hours later, this medium was replaced by a chemically defined medium. Dulbecco's modified Eagle's (DME)-F12 was used alone or supplemented with various combinations of transferrin, sodium selenite, or Ultroser G. The evolution of the culture and the islet ultrastructure were similar in defined and serum-containing media. However, in the defined medium, the neoformed islets seemed less numerous, and the fibroblast layer less dense, when compared to the RPMI+10% FBS control medium. At Day 7, in defined media, the total insulin content per dish was half that of control cultures. None of the tested additives improved the yield of the cultures. The fractional insulin release per day was elevated in defined media. In subsequent incubations, glucose and leucine stimulated insulin release in a way characteristic of these cells of fetal origin. The labeling index of islet cells cultured in DME-F12 reached 10.7%, which is not far from that observed in RPMI+10% FBS. Such a defined medium is useful to study B cell physiology, avoiding the possible interaction of serum components with substances to be tested. The support of the Fonds National de la Recherche Scientifique and the ‘Loterie Nationale’ of Belgium is also acknowledged.     

Supplementation of Ham's F10 culture medium with three different sera in the culturing of baboon oocytes

1. Ten female baboons (Papio ursinus) were stimulated for a total of 20 cycles with 3 ovulation induction agents. 2. Oocytes obtained were randomly allocated to Ham's F10 culture medium supplemented with human fetal cord serum, primate serum or commercial fetal bovine serum respectively. 3. Fertilization occurred (38.1-45.5%) in all 3 supplements, but cleavage and embryo development was more successful in culture medium supplemented with fetal bovine serum. 4. Eight embryos were cultured and 6 (75%) of these were cultured in fetal bovine serum supplemented medium.     

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.     

Anchorage-independent growth of normal human mesothelial cells: A sensitive bioassay for EGF which discloses the absence of this factor in fetal calf serum

Summary This laboratory recently reported that normal human mesothelial cells require epidermal growth factor (EGF) and hydrocortisone (HC), in addition to fetal calf serum and a complex defined medium component, in order to grow optimally in surface culture (9). We report here that this normal cell type also forms large colonies at high efficiency in semi-solid medium, but exhihits more stringent serum and EGF requirements for anchorage-independent than for surface growth. Mesothelial cells are unable to divide at all in semi-solid medium with added EGF or with less than 2% serum, whereas they grow slowly but progressively in surface culture under such conditions. In semi-solid medium containing 20% serum and HC, mesothelial cells are stimulated to divide by the addition of as little as 30 pg/ml purified EGF. Human urine or male mouse plasma could substitute for purified EGF, yielding growth commensurate with the levels of EGF in these biological fluids previously measured by others using radioreceptor and radioimmune assays. Thus growth of mesothelial cells in semi-solid medium can serve as a highly sensitive assay of EGF biological activity which is unaffected by the presence of serum proteins. In addition, our results demonstrate that fetal calf serum does not provide mitogenic levels of EGF to cultured cells, raising the question of the identity of plasma and serum mitogens. This work was supported by NIH grants RO1 AG02048 and RO1 CA26656 to James G. Rheinwald and by NIH postdoctoral fellowship F32 AG05303 to Paul J. La Rocca.     

Efficient Expression of a Transfected Foreign Gene by Cos1 Cells in Serum-free Medium

In this report, we have found that fatty acid-free bovine serum albumin stimulated the expression of a transfected foreign gene in Cos1 cells in serum-free medium and that its activity was as same as that of fetal calf serum. This will simplify the purification of the gene product from the culture medium.     

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.     

Differentiation of lymphoid cells. A selective anti-mitogenic component of normal serum which inhibits the induction of immunoglobulin production.

Rabbit lymph node cell populations cultured in vitro in the presence of fetal calf serum are induced to produce immunoglobulin M-secreting cells. The induction of such immunoglobulin production, measured by the capacity of the cell population to secrete immunoglobulins, was inhibited when cells were cultured with sera from a variety of species despite the presence of fetal calf serum. The addition of such inhibitory serum 36 hours after initiation of the cell culture or thereafter was without effect on the extent of induction of immunoglobulin production. On the other hand, the presence of inhibitory serum in culture during only the first 24 hours yielded the same inhibition as when serum was present throughout the 72-hour culture period. Inhibitory sera also suppressed the incorporation of thymidine into DNA. The induction of immunoglobulin production and the incorporation of thymidine into DNA were essentially equally inhibited by the same range of serum concentrations. Unlike conventional inhibitors of DNA synthesis, the inhibitory sera exhibited selective specificity with regard to the kind of cells that could be affected. Thus, such sera inhibited the DNA synthesis of lymph node cells cultured in the presence of fetal calf serum but did not inhibit concanavalin A-stimulated DNA synthesis of such cultured cells and, similarly, serum did not inhibit DNA synthesis of thymus cells cultured in the presence of fetal calf serum. The sera of all species examined were inhibitory except for fetal sera. As judged from a quantitative assay, bovine and porcine serum contained the highest titer of inhibitor, whereas sera from human, rat, mouse, and rabbit were clustered in a group exhibiting less inhibitor. Ascites fluid and lymph node extracellular fluids contained less inhibitor than found in the serum of the same animal and lysates of washed lymph node cells were devoid of inhibitor. Although fetal bovine serum and newborn bovine serum did not contain the inhibitor, it was detectable within 24 hours of parturition. The inhibitor is of relatively large apparent molecular weight (about 300,000) and has been purified about 70-fold.     

Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos

Bovine blastocysts were produced using 6 different systems: 5 commonly used in vitro culture systems (synthetic oviduct fluid medium - SOF- without fetal calf serum, SOF supplemented with 10% serum for the entire culture period, SOF supplemented with 10% serum from Day 4 of culture, M199 coculture with bovine oviduct epithelial cells, M199 coculture with granulosa cell monolayer) and 1 in vivo culture system involving collection of blastocysts from superovulated bovine donors at Day 7. Zygotes obtained from IVM/IVF were assigned randomly to 1 of the 5 systems tested and were cultured for 9 d (Day 0= day of insemination). Cleavage, development to the blastocyst stage and blastocyst sex ratio were assessed in all treatments. In addition, the effect of the IVC system on the kinetics of blastocyst development and sex ratio was assessed on Days 6, 7, 8, and 9. The presence of fetal calf serum in SOF not only resulted in faster development (19.1% of blastocysts in SOF supplemented with serum vs 7.1% in absence of serum at Day 6; P < 0.05) and increased blastocyst production (47.5% of blastocysts in SOF supplemented with serum vs 34.4% in absence of serum; P < 0.05) but it also enhanced overall male survival. The coculture systems produced fewer blastocysts than culture in SOF (27.6 to 28.3% in coculture vs 47.5% in SOF supplemented with serum; P < 0.05), but similar to SOF without fetal calf serum, they had no effect on blastocyst sex ratio.