Human Fibroblasts In Tissue Culture Research Articles

Quantitative Model of Cell Cycle Arrest and Cellular Senescence in Primary Human Fibroblasts

Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative “senescent” state. At early population doublings (PD), fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P) via reversibly cell cycle arrested (C) to irreversibly arrested senescent cells (S). In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.

The effect of nano- and micron-sized particles of cobalt–chromium alloy on human fibroblasts in vitro

Wear debris from metal on polyethylene joint replacements causes asceptic loosening as a result of an inflammatory reaction of macrophages to micron-sized particles. Metal on metal implants, which generate nanoparticles, have been reintroduced into surgical practise in order to avoid this problem. There is a current concern about possible long-term effects of exposure to metal particles. In this study, the cytotoxic and genotoxic effects of nanoparticles and micron-sized particles of cobalt chrome alloy have been compared using human fibroblasts in tissue culture. Nanoparticles, which caused more free radicals in an acellular environment, induced more DNA damage than micron-sized particles using the alkaline comet assay. They induced more aneuploidy and more cytotoxicity at equivalent volumetric dose. Nanoparticles appeared to disintegrate within the cells faster than microparticles with the creation of electron dense deposits in the cell, which were enriched in cobalt. The mechanism of cell damage appears to be different after exposure to nanoparticles and microparticles. The concept of nanotoxicology is, therefore, an important consideration in the design of future surgical devices.

Cell-surface binding sites for high density lipoproteins do not mediate efflux of cholesterol from human fibroblasts in tissue culture.

The present investigation was designed to test the hypothesis that binding sites for high density lipoproteins (HDL3) on cell surfaces of peripheral tissues mediate cholesterol efflux from these cells. This hypothesis had been formulated to explain two observations: 1) HDL3 binding to peripheral cells and HDL3-mediated cholesterol efflux from these cells had both been found to saturate at similar unbound (free) HDL3 concentrations; and 2) both of these processes had been found to be similarly "up-regulated" by loading the cells with cholesterol. In the present study, however, we found that the "specific" binding of HDL3 to cholesterol-loaded human fibroblasts was saturated at a free HDL3 concentration of approximately 20 micrograms protein/ml, whereas efflux of cholesterol from these cells to HDL3 did not "saturate" even at a free HDL3 concentration of 2000 micrograms protein/ml. In addition, we found that the increase in cholesterol efflux caused by loading the fibroblasts with cholesterol was no greater when the acceptor particles were HDL3 than when albumin or phospholipid vesicles served as acceptors, despite a marked increase in HDL3 binding to these cells. Because HDL3 binding to these cells and HDL3-mediated cholesterol efflux from these cells do not saturate at similar free HDL3 concentrations, and because the cholesterol-induced increase in HDL3 binding is not accompanied by a similar increase in cholesterol efflux that is specific for HDL3, we conclude that the described HDL3 binding sites on human fibroblasts do not mediate cholesterol efflux.

Radiosensitization of Human Fibroblasts by 3-Aminobenzamide: An Inhibitor of Poly(ADP-Ribosylation)

The poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide (3AB) increases the radiosensitivity of normal human fibroblasts in tissue culture in a dose-dependent manner at noncytotoxic concentrations. 3AB was also found to inhibit the repair of potentially lethal damage as well as the recovery occurring when radiation was delivered as fractionated vs single exposures. However, repair as measured by an increased survival when radiation is delivered at low dose rate compared to high dose rate was not effected by 3AB administration.

Direct in vitro effect of a sulfonylurea to increase human fibroblast insulin receptors.

We have studied the effects of the oral sulfonylurea agent glyburide to modulate insulin receptors on nontransformed human fibroblasts in tissue culture. When glyburide was added to monolayers of human fibroblasts, a dose-dependent increase in the number of cell surface receptors was observed with a maximum effect (19% increase) seen at 1 microgram/ml glyburide. Insulin can induce a loss of insulin receptors in these cells, and when fibroblasts are exposed to 100 ng/ml insulin for 6 h, approximately 60% of the initial complement of cell surface receptors are lost. When the process of insulin-induced receptor loss (or down regulation) was studied in the presence of glyburide, the drug exerted a marked inhibitory effect on this regulatory process. Thus, glyburide inhibited insulin-induced receptor loss in a dose-dependent fashion, and the maximally effective drug concentration (1 microgram/ml) inhibited 34% of the receptor loss. These studies demonstrate a direct in vitro effect of this oral hypoglycemic agent to increase the number of cell surface insulin receptors or prevent their loss, presumably by slowing the rate of receptor internalization. These findings may explain the well known extrapancreatic effect of sulfonylurea agents to improve insulin-mediated tissue glucose metabolism.

Synergistic effect of thrombin and platelet extract on growth of human fibroblasts

Earlier I found that thrombin stimulates proliferation of human fibroblasts in serum-free medium. This work demonstrates that thrombin, platelet extract and insulin have synergistic effect on growth of human fibroblasts in tissue culture and that addition of platelet extract but not of thrombin to medium containing 10 % of serum further increases the stimulatory effect. Raising the concentration of serum above 10 % did not markedly improve the growth promoting activity of the medium in spite of the obvious increase in the content of platelet factor.

Photoreactivation of thymine dimers in UV-irradiated human cells: Unique dependence on culture conditions

UV-irradiated human fibroblasts in tissue culture were exposed to photoreactivating light in an attempt to demonstrate a light-dependent loss of thymine dimers from the acid-insoluble fraction of the DNA. The only experimental conditions in which this phenomenon was observed was if the cells were grown for at least 10 days in Dulbecco's modified Eagle's minimum essential medium. Such cells lost a maximum of between 10–30% of the thymine dimers from their DNA during illumination for 1 h. When cells were grown in a variety of other media the phenomenon was not observed. The present experiments do not discriminate between true enzymatic photoreactivation and a medium-dependent photosensitization phenomenon that is not enzymatic in nature.

Inhibition by trimethoprim of some effects of epidermal growth factor on human fibroblasts in tissue culture

Inhibition by trimethoprim of some effects of epidermal growth factor on human fibroblasts in tissue culture

Putrescine transport is greatly increased in human fibroblasts initiated to proliferate.

Putrescine (diaminobutane) was previously found to stimulate proliferation of human fibroblasts in tissue culture, and a growth factor produced by these cells was identified as putrescine. In the present paper putrescine transport is studied. The rate of putrescine transport was dependent on temperature, and most of the labeled putrescine was retained by the cells after washing with excess unlabeled putrescine. The concentration of radioactivity after a [14 C]putrescine pulse was 85 times higher in the cells than in the medium, and over 95% of the radioactivity in the cells was as unchanged putrescine. Butanol treatment removed 70% of the radioactivity from the cells. The calculated Km was about the same for rapidly growing and for starved cultures, while Vmax was higher for the former than for the latter cultures. Putrescine transport was inhibited to varying degrees by other polyamines, but not by amino acids or divalent cations. Stimulation of cell proliferation by serum was followed by an 18-100-fold increase in the rate of putrescine transport, which was not inhibitable with cyclic AMP, dibutyryl cyclic AMP, or prostaglandin E1. Removal of serum resulted in a rapid decrease in the rate of putrescine transport. Insulin in low serum medium and trypsin in the absence of serum also accelerated putrescine transport. Moreover, the rate of putrescine transport was dependent on cell density. It was faster in sparsely populated than in densely populated cultures. SV40-transformed human fibroblasts responded to addition and removal of serum in the same way as the untransformed parent cell line.

Metabolism of L-thyroxine (T4) and L-triiodothyronine (T3) by human fibroblasts in tissue culture: evidence for cellular binding proteins and conversion of T4 to T3.

The metabolism of L-thyroxine (T4) and L-triiodothyronine (T3) by human fibroblasts in tissue culture was studied. Fibroblasts were incubated in serumless medium and in media containing various concentrations of normal human serum (HS) or fetal calf serum (FCS) enriched with both T4-125I and T3-131I, and the simultaneous cell uptake of both tracers and the T4 to T3 conversion determined. Interference from the preferential cellular uptake of T3 impurities in T4 was corrected by determination of the 125I/131I ratio migrating as T3 on bidimensional paper chromatography of medium and cells. In the absence of serum in the medium, cell to medium (C/M) concentration gradient was 90 to 100 for T3 and 49 to 58 for T4. With increasing amounts of HS, the fractional uptake of T3 and T4 diminished and the C/M gradient for T4 was less than 1. The cellular T3/T4 ratio increased to a maximum of 11.4 in the presence of 5% HS. The T3/T4 ratio in the dialysable fraction was 1.01 for serumless medium and 5.8 for medium conta...

Synthesis of cobalamin coenzymes by human cells in tissue culture.

Abstract Humans, as well as other mammals, must convert ingested vitamin B 12 to active coenzyme forms for biologic activity. We found that human fibroblasts in tissue culture are capable of carrying out this conversion and we report a method to measure the accumulation of newly synthesized coenzymes by the cultured cells. Fibroblasts are grown in culture medium which contains the precursor vitamin, 57 Co-hydroxocobalamin. After 4 days the cells are harvested. Cobalamins are extracted from the cells and then separated by thin layer chromatography. In addition to the hydroxocobalamin, two peaks of radioactivity are found which correspond to the coenzyme forms of B 12 , deoxyadenosylcobalamin and methylcobalamin. The ability to study coenzyme synthesis and accumulation in human cells will enable investigation of normal B 12 metabolism and of human disease states where abnormal metabolism is suspected.

54. Binding capacity of human albumin for bilirubin

The binding between bilirubin and human serum albumin was studied spectrophotometrically and with Sephadex gel filtration. Spectral absorption curves of solution of bilirubin in buffers containing decreasing amounts of albumin were registered. At a molar bilirubin to albumin ratio of 1:1 a change in these curves takes place, indicating that only one molecule of bilirubin is tightly bound to albumin. Sephadex gel filtration studies also showed that with solution containing bilirubin and albumin in molar ratios above 1:1 bilirubin seemed, however, to be more loosely bound.The toxic effect of bilirubin on human fibroblasts in tissue culture was also tested. When solutions containing bilirubin to albumin ratios above 1:1 were added to the growth medium, a toxic effects was seen on the fibroblast growth. With a bilirubin to albumin ratio of 2:1 rapid cell death was found with total bilirubin concentrations as low as 5 mg/100 ml. On the other hand, when the bilirubin to albumin ratio in the growth medium was below 1:1, the cells tolerated bilirubin concentrations as high as 20 mg/100 ml, which was the highest tested. It therefore seems that only one molecule of bilirubin is tightly bound to each molecule of albumin, ab\nd that this molecule only is detoxified.

Some effects of diazepam on the fine structure of human fibroblasts in tissue culture

A study of the effects of diazepam on the fine structure of human fibroblasts in tissue culture is presented. Cells were grown in a culture medium containing diazepam in concentrations of 0.1, 1.0, 10.0, and 20.0 μg per milliliter and were compared with cells unexposed to the drug. Cultures were prepared for study by light and electron microscopy. No significant changes in cellular morphology were noted at the light microscopic level. At ultrastructural levels, alterations in organization of the cytoplasm, which appear to be dose dependent, were noted. These alterations were most prominent in the membranous elements of the cell and consisted of the appearance of increased numbers of large and small vesicular structures, including many bizarre forms. Disruption of elements of smooth and rough endoplasmic reticulum, as well as areas of focal cytoplasmic degeneration, was also seen.

Electron microscopy of human fibroblasts in tissue culture during logarithmic and confluent stages of growth

The structure of human fibroblasts in the logarithmic and in the stationary phase of growth was examined by electron microscopy. The most striking changes in the fibroblasts as they passed into confluency were the dilatation of the endoplasmic cisternae, the loss of the polyribosome configuration, and the increase in the number of microfilaments. More subtle changes were the increase in the amount of condensation of the nuclear chromatin, and an increase in the number of autophagic vacuoles.

Evidence against the existence of guanosine and inosine kinases in human fibroblasts in tissue culture

Human HGPRT- cells derived from patients with the Lesch-Nyhan syndrome have been labeled with 3H-hypoxanthine, 3H-guanosine and 3H-inosine in an effort to demonstrate inosine kinase or guanosine kinase activity. HGPRT- fibroblasts showed virtually no incorporation of any of these labeled nucleic acid precursors into TCA insoluble material when the cells were examined by autoradiographic techniques. We interpret these results as indicating either that these tissue culture cells have no detectable inosine kinase or guanosine kinase activity or that although the kinases are present they are also inactive due to a defect in a polypeptide subunit or ligand common to HGPRT and the kinases.

Metabolic Cooperation between Genetically Marked Human Fibroblasts in Tissue Culture

GENETIC variants of several mammalian cell lines which are not able to incorporate preformed purine or nucleoside into the cell's nucleic acids have been known for some time. In some of these cell lines a lack of activity of the enzyme inosinic pyrophosphorylase (IPP), also called hypoxanthine-guanine phosphoribosyltransferase (PRT) (EC 2.4.2.8), has been demonstrated1–4. This enzyme catalyses the conversions both of hypoxanthine to inosinic acid and of guanine to guanylic acid, and constitutes an essential part of one of the purine “salvage” pathways. Seegmiller and his colleagues5 have recently shown that the severe sex-linked neurological disease of humans known as the Lesch–Nyhan syndrome is associated with the virtual complete absence of this enzyme activity from brain and all other tissues which have been examined6.

INHIBITION OF VARICELLA VIRUS BY 5-IODO-2'-DEOXYURIDINE.

SummaryAlthough 5-iodo-2′-deoxyuridine (5-IDU) has some effect on the multiplication of human fibroblasts in tissue culture, it also markedly prevents cytopathic effects of varicella-zoster virus even when added to the tissue culture in concentrations of 5 μg/ml as long as 3 days after challenge with the virus. The compound will inhibit further plaque formation even after cytopathic effects are already evident. Production of plaque-forming virus particles is likewise suppressed by the compound. These findings provide indirect evidence that varicella-zoster virus is a DNA virus, and they suggest that perhaps this compound might find some use in cases of serious human disease produced by this virus.