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 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.     

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 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.     

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.     

Heat evolution by human skin fibroblasts in monolayer culture

Human skin fibroblasts were attached to a plastic foil and cultivated in a batch calorimeter. The cultures exhibited a characteristic heat profile, whose different phases were attributed to the processes of spreading and growth of the seeded cells, to cell multiplication and to the metabolism of maintenance. An enthalpy change of (1.5 ± 0.3) μJ per dividing cell and, during confluency, a heat production of (40 ± 10) pW/cell were determined. The monolayer cultures could be kept alive and free of microbial contamination for more than 5 days within the calorimetric vessel.     

Growth of human skin fibroblasts in dialyzed fetal bovine serum

Summary Human diploid fibroblast cultures plated at or below a density of 2×10³ cells per cm² grew very slowly or not at all in MEM supplemented with 10% fetal bovine serum that had been dialyzed for 24 hr. Adding serine (0.2 mM) or pyruvate (1.0 mM) to MEM and 10% dialyzed serum restored growth to the level observed with 10% nondialyzed serum. Serine and pyruvate also were able to overcome partially the growth arrest induced by a reduced serum concentration (1 or 2%). Human fibroblast cultures grew very well in 100% fetal bovine serum that had been dialyzed against MEM. For cells grown in dialyzed serum, the final number increased with increasing serum concentration, in contrast to the well established toxic effects of high concentrations of nondialyzed serum. This research was supported by NIH Grants CA15207 and HD03110.     

The use of human fibroblasts grown on microcarriers for the formation of the connective tissue equivalent

Live equivalents of tissues, specifically those produced on the basis of fibroblasts and collagen gel, are widely used for repair of organ and tissues defects. In clinical practice, it is more convenient to use the fibroblasts grown on microcarriers or such a connective tissue equivalent when the fibroblasts on microcarriers are embedded in collagen gel. We studied the properties of a connective tissue equivalent produced by embedding the fibroblasts grown on microcarriers in collagen gel for its prospective use in clinical practice. According to our results, the optimal time of use of the live tissue equivalent amounts to three--four days after embedding of fibroblasts on microcarriers in gel. At that time, contraction only begins, which facilitates manipulations with the gel.     

Biosynthesis and release of glycoproteins by human skin fibroblasts in culture

1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-³H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated ³H was present as [³H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [³H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [³H]fucose-labelled material before and after trypsin digestion. 3. The [³H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000–250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [³H]fucose and ¹⁴C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [³⁵S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.     

Influence of cell culture conditions on aromatase activity in human genital skin fibroblasts

Summary Because the measurement of aromatase activity in cultured human genital skin fibroblasts has been proposed as a means of studying estrogen production in men, we investigated the influence of culture conditions on aromatase activity. Genital skin fibroblasts were seeded onto culture plates at a density of 1×10⁶ cells/plate and aromatase activity was determined over a 1-mo. period. Enzyme activity rose slowly over the first 14 d but then rose rapidly to a 10-fold higher plateau by Day 28. The rise in aromatase activity was similar whether activity was normalized for protein or for DNA content. When cells were seeded at the usual density of 1×10⁶ or at 0.25×10⁶ cells/plate, aromatase activity was consistently lower during the first 2 wk in cells plated at lower density, but thereafter the levels of enzyme activity in the two groups converged. In cells plated at the lower density, the lower activity observed in the first 2 wk was associated with a lower V _max . Preincubation of cells plated at one density with conditoned medium from cells plated at the other density did not change the relatve levels of activity in the two groups. By contrast, dihydrotestosterone (DHT) receptor binding and 5α-reductase activity were similar at all time points, despite differences in plating density. In additional experiments, the culture medium was replaced daily rather than every 3rd d, and aromatase activity was assayed on Day 7. In cells fed daily, DNA and protein content were twice that of cells fed every 3rd d. By contrast, aromatase activity declined to 30% of the in the latter group. DHT and dexamethasone receptor binding and 5α-reductase activity were similar in the two groups. In summary, factors such as plating density, culture density, and frequency of media replacement dramatically influence aromatase activity in cultured human genital skin fibroblasts. Therefore, the interpretation of aromatase activity data obtained from cultured cells in relation to physiologic or pathologic states should be viewed with appropriate caution. The work was supported in part by grants R01 DK 35339 and R01 DK 00180 from the National Institutes of Health, Bethesda, MD, and by RR 00035 from CLINFO Systems at the Johns Hopkins University School of Medicine, Baltimore, MD.     

Effect of testicular hyaluronidase on hyaluronate synthesis by human skin fibroblasts in culture

The effect of hyaluronidase treatment on the incorporation of [3H]glucosamine into hyaluronate in human skin fibroblast cultures was investigated. Fourth passage cells in confluent cultures were treated with hyaluronidase from bovine tests, Streptomyces and leech in Dulbecco's minimum essential medium in the presence of 3% fetal calf serum. The medium was removed from the control (non-treated) and the treated cultures and the washed cell layers were incubated with [3H]glucosamine and [35S]sulfate. [3H]Hyaluronate was separated by DEAE Trisacyl chromatography and identified by specific enzymic assays. Hyaluronidase treatment induced an increase in the amount of labelled hyaluronate secreted into the medium and into the pericellular compartment. This amount reached a plateau with increasing enzyme concentration and with the time of treatment. Oligosaccharides derived from hyaluronate did not produce this effect. The maximal increase was about 3-fold, and was not inhibited by exogenous hyaluronate (25-100 micrograms/ml) or by oligosaccharides from hyaluronate. Cycloheximide (0.03 mM) inhibited hyaluronate synthesis by 18% or less in the control cells and by 50% in the hyaluronidase-pretreated fibroblasts. No significant difference was found in the hyaluronate synthase activity between control and treated cells, at 60 min following treatment, indicating the reversibility of the effect. The persistence of the stimulation required the presence of hyaluronidase. The treatment of cells with specific hyaluronidases (from Streptomyces and leech) or with testicular hyaluronidase did not modify the labelling of the sulfated glycosaminoglycans. The incorporation kinetics of the [3H]glucosamine into labeled hyaluronate and the increased amount of non-labelled hyaluronate determined by radiometric assay indicated a specific stimulation of hyaluronate synthesis in the hyaluronidase-pretreated fibroblast cultures.     

Effects of some antibiotics on the growth of human diploid skin fibroblasts in cell culture

Summary During serial subcultures 50 μg per ml gentamicin and penicillin (100 U per ml)-streptomycin (100 μg per ml) depressed cell growth significantly 2 weeks after the addition of the antibiotics; gentamicin, but not penicillin-streptomycin, stimulated cell growth before it became inhibitory. Removal of the antibiotics resulted in the cell yield returning to normal. The results show that these antibiotics can be harmful to cells even at concentrations thought to be safe.     

The presence of lipocortin in human embryonic skin fibroblasts and its regulation by anti-inflammatory steroids

Human embryonic skin fibroblasts in culture produce pro-inflammatory lipid mediators and all types of prostanoids. When these cells were treated with the anti-inflammatory steroid, dexamethasone, prostaglandin production was inhibited. This phenomenon required glucocorticoid receptor occupancy and mRNA and protein synthesis. The inhibitory effect was prevented by treating the cells with a monoclonal antibody, BF 26, raised against renocortin, a lipocortin-like protein formed in rat kidney medulla interstitial cells in culture. When the proteins present in the supernatants and the cell pellets derived from control and dexamethasone-treated cells were analyzed for their ability to inhibit phospholipase A2, four inhibitory peaks, at 45, 30, 15 kDa and one peak under 12 kDa, were found in the supernatants of control and dexamethasone-treated cells, whereas one single inhibitory peak at 15 kDa was found in the cell pellets. The antiphospholipase activity was much greater in dexamethasone-treated cells than in control cells. These results suggest that preformed lipocortin exists in human cells and that lipocortin is synthesized and released under glucocorticoid treatment.     

Electron microscopy of human skin fibroblasts in situ during growth in culture

We have studied the electron microscopic (EM) appearance of cultured diploid human skin fibroblasts during logarithmic and confluent stages of growth using a simple technique which permits in situ visualization of individual cells in monolayer. By comparison, cells disrupted from a monolayer and pelleted showed drastic distortion of the cell surface and appearance of organelles: mechanical scraping produced massive dilatation of the rough endoplasmic reticulum (RER); and trypsin-produced multiple blebs of the plasma membrane and cytoplasmic vacuoles. In situ, these changes in trypsinized cells disappeared within 1 h after plating. Six hours later, microtubules and microfilaments had surrounded the nucleus and were oriented in longitudinal bundles beneath the plasma membrane during rapid growth. At confluence these cytoskeletal elements seemed to extend beyond the plasma membrane at intercellular junctions. Pinocytotic vesicles were abundant at those surfaces devoid of filaments. Mitochondria, aligned with the long axis of the cell, were extremely long and narrow. During logarithmic growth there were many free ribosomes, polysomes, and some flat cisternae of RER. At confluence most ribosomes were found in spirals on dilated saccules of RER which contained electron-dense material. Lysosomes of several types were present during all phases of growth and varied in number from cell to cell. Late in culture the lysosomes tended to be larger, often occupying whole areas of cytoplasm or even extruding from the cell, and resembled those seen in lysosomal storage diseases. Understanding the in situ ultrastructure of normal human fibroblasts during growth in culture will permit systematic examination of such cells in a variety of pathological states.     

Alkaline phosphatase activation and collagen synthesis in human skin fibroblasts in culture

It has been noted in regenerating wounds that alkaline phosphatase activity in fibroblasts reaches a maximum when the collagen production is greatest. Tissue culture studies were carried out to show that prednisolone phosphate, while increasing the specific activity of alkaline phosphatase in human diploid skin fibroblasts, did not affect accumulation of collagen-hydroxyproline in monolayers or media. Addition of sodium ascorbate, resulted in rapid accumulation of hydroxyproline in the culture over a 13-day interval, while alkaline phosphatase activity increased only slightly over the last 6 days. When prednisolone and ascorbate were added in combination, alkaline phosphatase activity was significantly increased: but accumulation of hydroxyproline was no greater than in cultures to which ascorbate alone was added. Activation of alkaline phosphatase induced by prednisolone phosphate does not appear to be directly related to the biosynthesis of collagen in human skin fibroblasts in tissue culture.