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.     

Vascular endothelial cells: Targets for studying the activity of hair follicle cell-produced VEGF

Fetal bovine aortic endothelial cells (FBAEC) were exposed to purified fractions of conditioned medium from cultures of hair dermal papilla cells (DPC) to determine the existence of any vascular endothelial growth factor (VEGF)-like paracrine activity of the latter. Such fractions were tested for stimulation of growth and migration of cultured FBAEC. In addition, VEGF secretion by DPC was measured by radioassay of VEGF receptors using FBAEC as target cells. The results showed that stimulation of FBAEC proliferation and migration following exposure to purified conditioned medium was dose-dependent. Radioreceptor assays of recombinant VEGF and purified DPC-conditioned medium showed competitive VEGF binding in FBAEC.Abbreviations CM conditioned medium - DMEM Dulbecco's modified eagle's medium - DPC dermal papilla cells - EDTA ethylenediaminetetra-acetic acid - FBAEC fetal bovine aortic endothelial cells - FCS fetal calf serum - VEGF vascular endothelial growth factor     

An Improved Chemical Substitute for Fetal Calf Serum for the Micronucleus Test

The routine use of the micronucleus test in the mutagenicity evaluation of xe-nobiotics is limited by high cost and limited availability of fetal calf serum. On the other hand, there are disadvantages, such as hypotonic damage and clumping of cells, associated with the use of mineral medium substitutes for fetal calf serum. Alternatively, we recommend a chemical medium containing Hanks' buffered salt solution, 1% (w/v) bovine serum albumin, and 0.15% (w/v) ethylenediaminetetraacetic acid, final pH 7.4, to preserve morphology, density and homogeneity of bone marrow cells. Mast cell granules are efficiently removed from rat bone marrow preparations by washing twice with this medium. The morphological preservation of cells is further enhanced by fixation with 70 % (v/v) ethanol for 5 min. The proposed medium provides a cost-effective and convenient substitute for fetal calf serum with substantially improved quality of bone marrow preparations for the micronucleus test.     

Angiotensin-converting enzyme: accumulation in medium from cultured endothelial cells.

The activity of angiotensin converting enzyme has been measured in endothelial cells cultured from hog aorta, and in serum-free culture medium taken from both endothelial cells and smooth muscle cells. Endothelial cells maintained in medium containing 20% fetal calf serum contained 43 pmol/min/10⁶ cells of converting enzyme activity; freshly collected cells contained 518 pmol/min/10⁶ cells. Endothelial cells held in serum-free medium release this activity into the medium in amounts up to 40 times that associated with the cells; at the same time the activity associated with the cells rises 2 fold. The rise in cell-associated activity and the appearance of activity in the medium are both blocked by cycloheximide. These observations provide direct evidence that endothelial cells can produce excess angiotensin-converting enzyme and release it in active form, and thus serve as the source of circulating converting enzyme activity.     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

Role of thrombospondin in the adhesion of human endothelial cells in primary culture

Summary The role of thrombospondin on the adhesion of endothelial cells in primary culture was studied using a serum-free defined medium or thrombospondin-depleted fetal bovine serum. Under these conditions, only 6% of the cells adhered to gelatin-coated dishes, whereas cells adhering to gelatin in the presence of normal fetal bovine serum were considered as 100% adhesion. The percentage of cells attached to fibronectin or thrombospondin-coated dishes in thrombospondin-depleted serum was 66 and 32%, respectively. The addition of purified platelet thrombospondin to thrombospondin-depleted serum increased the adhesion of endothelial cells to gelatin and to thrombospondin, up to 32 and 59%, respectively, and restored the attachment to fibronectin to the same extent as that observed in the presence of normal serum. In contrast to the attachment, the spreading of the adhering cells was not further influenced by the addition of soluble thrombospondin. Subcultured cells did not require any protein for adhering to gelatin substrata. These observations indicate that thrombospondin plays a major role in the adhesion of endothelial cells in primary culture.     

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.     

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.     

Reversible alterations in cultured pulmonary artery endothelial cell monolayer morphology and albumin permeability induced by ionizing radiation

The effects of ionizing irradiation (0, 600, 1,500, or 3,000 rads) on the permeability of pulmonary endothelial monolayers to albumin were studied. Pulmonary endothelial cells were grown to confluence on gelatin-coated polycarbonate filters, placed in serum-free medium, and exposed to a 60Co source. The monolayers were placed in modified flux chambers 24 hours after irradiation; 125I-albumin was added to the upper well, and both the upper and lower wells were serially sampled over 4 hours. The amount of albumin transferred from the upper well/hour over the period of steady-state clearance (90-240 min after addition of 125I-albumin) was 2.8 +/- 0.2% in control monolayers and was increased in monolayers exposed to 1,500 or 3,000 rads (increase of 63 +/- 10% and 61 +/- 10%, respectively, P less than 0.01). No increase was found in monolayers exposed to 600 rads. The increases in endothelial albumin transfer rates were associated with morphologic evidence of monolayer disruption and endothelial injury which paralleled the changes in albumin permeability. Dose-dependent alterations in endothelial actin filament organization were also found. Incubation of the monolayers exposed to 3,000 rads with medium supplemented with 10% fetal calf serum for 24 hours resulted in normalization of albumin permeability, improvement in morphologic appearance of the monolayers, and reorganization of the actin filament structure. These studies demonstrate that ionizing radiation is an active principle in the reversible disorganization of cultured pulmonary endothelial cell monolayers without the need of other cell types or serum components.     

Cellular adhesion to collagen

BALB/3T3 cells were released from tissue culture plates with EGTA, and their rates of attachment to collagen gels polymerized on Millipore filters; were measured. Cell attachment in serum-free medium was 20–50% of that which occurred in medium containing 10% fetal calf serum (FCS). Cell attachment to gels pretreated with FCS and assayed in serum-free medium was identical with that of gels in FCS-containing medium. Thus, it seems there are two separate mechanisms of attachment to collagen; one involving direct attachment and a second mediated by a serum component(s) which binds to collagen.     

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.     

Extraction of an erythrotropin-like factor from bovine serum albumin (cohn fraction V)

Summary Different batches of commercially available bovine serum albumin (Cohn fraction V) were tested in a serum-free medium for their ability to stimulate thymidine incorporation in erythroid cells of fetal bovine liver. All preparations stimulated thymidine incorporation. Crystallized, charcoal-treated, or fatty acid-free albumin had substantially lower thymidine incorporation-stimulating activities than the crude preparations. The albumin preparations also had a synergistic effect with respect to erythropoietin on erythroid cells from rat liver, a typical property of erythrotropins. One gram of one of the batches of Cohn fraction V was fractionated by reversed-phase high performance liquid chromatography (HPLC). The fraction with thymidine incorporation-stimulating activity had a similar elution position as erythrotropin isolated from fetal bovine serum. Further purification using reversed-phase HPLC in the presence of trifluoroacetic acid and heptafluorobutyric acid and gel permeation HPLC resulted, in the isolation of a factor that is very similar to fetal bovine serum erythrotropin. It has practically the same specific activity as the purified fetal peptide in the rat liver bioassay. These results suggest that many of the beneficial effects of the albumin preparations added as supplement of serum-free tissue culture media may be due to the presence of erythrotropin-like factors. The work was supported by grants MT-6072 and ME-9031 from the Medical Research Council of Canada. The author is a Chercheur-Boursier of the Fonds de la Recherche en Santé du Quebec.     

Demonstration of the specific binding of bovine transferrin to the human transferrin receptor in K562 cells: evidence for interspecies transferrin internalization

Specific binding of ferric bovine transferrin to the human transferrin receptor was investigated using K562 cells propagated in serum-free medium without transferrin supplemented with 10(-5) elemental iron. Affinity chromatography of solubilized extracts of K562 cells surface-labeled with 125I was performed using bovine transferrin- and human transferrin-Sepharose 4B resins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of resin eluates reveal that bovine transferrin specifically binds a Mr = 188,000 protein which dissociates into a Mr = 94,000 protein under reducing conditions, a finding identical to what is seen with human transferrin. The Mr = 94,000 reduced protein isolated by bovine transferrin resin shows an identical one-dimensional partial proteolytic digestion map with that of the human transferrin receptor. Unlabeled bovine transferrin was shown to specifically compete 125I-labeled human transferrin from the human transferrin receptor on the surface of K562 cells at 4 degrees C in a similar manner as unlabeled human transferrin; however, approximately a 2,000-fold higher concentration of bovine ligand was required to achieve comparable competition (50% inhibition of binding). Indirect immunofluorescence cytolocalization of bovine transferrin in K562 cells grown in serum-free medium supplemented with ferric bovine transferrin reveal patterns similar to those seen for human transferrin (both focal perinuclear and diffuse cytoplasmic fluorescence). Monensin treatment results in a dramatic accumulation of bovine ligand in perinuclear aggregates, suggesting that it is recycled through the Golgi, as is human transferrin. K562 cells grown in serum-free medium supplemented with either 300 micrograms/ml of ferric human or ferric bovine transferrin were found to demonstrate superimposable growth curves.     

Rabbit chondrocytes maintained in serum-free medium : I. Synthesis and secretion of hydrodynamically-small proteoglycans

The biosynthesis of sulfated proteoglycan in vitro by rabbit articular chondrocytes in first passage monolayer culture maintained in fetal bovine serum (FBS) or in serum-free conditions was compared. Neosynthesized proteoglycan in the culture medium in the most dense fraction of an associative CsCl density gradient (fraction dAl) declined with increasing time under serum-free conditions, but not when cells were maintained in the presence of serum. After one day, the major peak of incorporated 35SO4 in medium fraction dAl eluted as a retarded peak (Kav 0.28) on Sepharose CL-2B, whether cells were maintained under serum-free or serum-containing conditions. The hydrodynamic size of proteoglycan monomer fraction dAlDl obtained after one day of exposure to serum-free culture media was smaller than dAlDl from serum-containing cultures. The hydrodynamic size of dAlDl obtained from serum-free culture media became even progressively smaller after 2 and 3 days' exposure to these conditions. Hydrodynamically small sulfated proteoglycans were identified in the cell-associated dAlDl fraction as early as one day after switching chondrocytes from serum-containing to serum-free medium. Culture medium fraction dAlDl from serum-free culture medium aggregated poorly when incubated with human hyaluronic acid (HA) in the presence of bovine link protein or when dialysed against bovine nasal cartilage proteoglycan aggregate. Proteoglycan monomer from serum-containing medium reaggregated more efficiently under both conditions. No change in the size of glycosaminoglycan chains was seen in the smaller proteoglycan subpopulations, nor was there any indication of marked changes in the glycosaminoglycan types.     

A low-serum medium with BSA and ferrous sulfate for the production of tissue plasminogen activator by human embryo lung cells

A low-serum medium containing bovine serum albumin (BSA) was investigated with respect to the growth of and tissue plasminogen activator (TPA) production by human embryo lung (HEL) cells on microcarrier beads and in collagen gel. BSA and ferrous sulfate were chosen as substitutes for fetal calf serum (FCS) through a simple screening test involving many substances. The growth promoting effects of BSA and ferrous sulfate were independent of each other and from the FCS concentration. Though BSA inhibited initial cell attachment to the carrier surface, it did promote the growth of cells attached to microcarrier beads. Cells grown on microcarrier beads in the low-serum medium containing BSA, ferrous sulfate and 3% FCS produced an amount of TPA similar to that produced by ones grown in the 10% FCS medium. Although cells on the dish surface did not grow at all on serum-free media containing BSA and ferrous sulfate, cells in the collagen gel were able to grow slightly on the serum-free medium. Cells grown on the low-serum medium in collagen gel produced more TPA over a long period than those in the microcarrier beads using the low-serum medium. The optimum concentration of proteose peptone in the TPA production medium for the collagen gel culture was similar to that for the dish surface culture.     

Oligodeoxynucleotide stability in subcellular extracts and culture media

Oligodeoxynucleotide degradation was studied in four systems in order to assess the importance of degradation in hybridization arrest experiments dependent on oligodeoxynucleotides complementary to mRNA sequences. Oligodeoxynucleotides were not detectably degraded over 2 h at 37 degrees C in rabbit reticulocyte lysate or Dulbecco's modified essential medium with 5% fetal calf serum, but were degraded over 2 h in HeLa cell postmitochondrial cytoplasmic extract, and were degraded within 15 min in bovine calf serum.     

Characterization of a serum factor that decreases albumin mRNA in cultured hepatocytes

Summary When primary cultures of hepatocytes are exposed to media containing fetal bovine serum (FBS) there is a rapid decrease in levels of tissue-specific mRNAs such as albumin mRNA. We used Northern blot analysis to examine mRNA levels in cultured hepatocytes, and characterized the factor in FBS that significantly reduces the steady state albumin mRNA level. Neonatal bovine serum or serum derived from platelet-poor calf plasma proved as potent as did FBS, but commercial bovine serum albumin did not exhibit this inhibitory activity. Inhibitory activity of FBS was not removed by moderate heat treatment, dialysis, or extraction with organic solvents. However, incubation of FBS with a highly anionic detergent such as 0.1% sodium dodecyl sulfate orN-lauroyl sarcosine, followed by extensive dialysis, resulted in sera that did not inhibit expression of albumin mRNA. These sera supported cell attachment and seemed non-toxic toward the cells. Ammonium sulfate fractionation of FBS showed the activity was present in the 45 to 70% fraction, and trypsin digestion destroyed the inhibitory activity. Gel exclusion chromatography gave a molecular weight 60 000 to 70 000. Fractionation of serum proteins by DEAE-Sephacel or Cibacron blue-agarose showed enrichment for albumin in the most active fractions. Interestingly, metabolic labeling of secreted and cellular proteins with³⁵S-methionine and cysteine showed no significant difference between hepatocytes maintained for 2 days beforehand in serum-free or serum-supplemented media, and no difference between detergent-treated FBS and control FBS. Therefore, FBS contains a factor that causes a significant decrease in steady state levels of mRNA for albumin and other mRNAs of tissue specific function, but under these conditions albumin mRNA levels are not paralleled by synthesis of albumin or other proteins.     

Immobilized serotonin: a novel substrate for cell culture

Baby hamster kidney cells, bovine aortal endothelial cells, bovine smooth muscle cells, and chick embryo fibroblasts were all observed to attach and grow on serotonin which had been immobilized by covalent coupling to agarose beads. While growth and morphology of cells on immobilized serotonin appeared normal, a change in cell function may have occurred since the pattern of polypeptides expressed by these cells was different from that of cells grown on two other substrates: immobilized fibronectin and tissue culture plastic. By changing the composition of the fetal calf serum proteins in the growth medium it was shown that cells attach directly to immobilized fibronectin without mediation by medium components. In contrast, cells were found not to attach directly to immobilized serotonin but to attach indirectly via factors absorbed onto immobilized serotonin from fetal calf serum. The major component of this cell attachment activity was shown not to be fibronectin and was identified following separation by SDS-PAGE, electroblotting, and cell binding on nitrocellulose filters. The cell attachment activity compromises a major protein species of Mr 70,000 which is the molecular size of the recently identified serum spreading factor also called vitronectin.