Interactions of B16F10 melanoma cells aggregated on a cellulose substrate.

There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

Culture of melanoma cells as aggregates on cellulose substratum.

Cells aggregate on an original cellulose substratum (CEL). This influences the signaling programs of adhering cells. CEL thus appears to be a suitable tool for studying the regulation of cell-substratum and cell-cell interactions.

Explants of porcine coronary artery in culture: A paradigm for studying the influence of heparin on vascular wall cell proliferation.

Explant cultures of porcine coronary artery provided a coculture model, used as a paradigm of arterial wall in contact with vascular prosthesis which allowed the study of spatial and temporal changes in cell phenotype. First cells emerging from the explant had an endothelial phenotype monitored by cytoimmunostaining. Percentages of anti-smooth muscle alpha-actin labelled cells were assessed at early and late phase by flow cytofluorometric analysis to control the effect of heparin. At 100 mug ml(-1), no effect on alpha-actin labelled cell growth has been detected. This result contrasted with the inhibition of monolayer cell cultures. At 500 mug ml(-1), the proliferation of smooth muscle cells was reduced. This explant system should be useful for testing drugs susceptible to interfere with restenosis.

cAMP levels in cells attached to AN69 and Cuprophan: cAMP dependence of cell aggregation and the influence of serum.

We have examined the link between the aggregation or spreading of cells adhering to substrata of differing biocompatibility and activation of the cyclic AMP (cAMP) pathway. We compared the rate at which the Mouse Swiss 3T3 fibroblasts attached to Cuprophan (CU), AN69 and a control plastic in the presence and absence of foetal calf serum (FCS). Serum had no effect on the kinetics of cell attachment to CU or AN69. Cells incubated in culture medium containing 10% FCS aggregated on CU, whereas they spread on AN69 and plastic. Aggregated cells contained significantly higher concentrations of cAMP than cells spreading, and aggregation was prevented by treatment with miconazole, an inhibitor of adenylyl cyclase. cAMP-dependent cell aggregation occurred on all three substrata in serum-free medium, suggesting that proteins adsorbed onto AN69 and plastic in the presence of serum helped protect the cells. Far less serum protein was adsorbed onto CU than onto AN69 or plastic, consistent with the similar increases in cAMP in cells attached to CU with or without serum.

Heparin immobilized on proteins usable for arterial prosthesis coating: growth inhibition of smooth-muscle cells.

Gelatin or a mixture of albumin and gelatin has been proposed for the coating of vascular grafts according to their surface thrombogenicity and biocompatibility, and the possibility of biodegradation. Heparin treatment of hemocompatible surfaces improved the patency of prostheses. In this study, different amounts of heparin were immobilized on these protein gels using a water-soluble carbodiimide [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide]. The results showed a coupling of heparin with gelatin and/or albumin at the surface of the gels, stable for as long as 1 month. From 0.20 to 3.60 microg x cm(-2), heparin could be immobilized. The antiproliferative activity of immobilized heparin was controlled toward bovine smooth-muscle cells grown on these gels. Cell growth inhibition was dose dependent, but the percentages of inhibition were lower at day 8 than at day 4 at any heparin concentration used under experimental conditions. Referring to heparin in solution, immobilized heparin displayed an antiproliferative activity that improved the potential interest for coating.

Cyclic AMP in cells adhering to bioincompatible (Cuprophan) and biocompatible (AN69) substrates.

The processing of signals produced when cells contact biomaterials was examined. Of the several possible pathways, this study focuses on the amount of cAMP that accumulated in NIH 3T3 cells during the first 45 min after the cells contacted the bioincompatible membrane Cuprophan (CU) and the biocompatible membrane AN69. The cells that adhered to CU contained more cAMP than those that attached to AN69. This might be because the cells did not spread but remained rounded up under scanning electron microscopy. There was no increase in cAMP in the cells that did not adhere to CU. The cAMP-modulating agents, forskolin and isoproterenol, were used to assess the cAMP-generating capacity of adenylylcyclase in cells adhering to CU and AN69. This capacity was not affected by a high concentration (100 microM) of forskolin. Isoproterenol had no effect on the cAMP content of the cells, demonstrating that beta adrenergic receptors are not implicated in the activation of cAMP production by membranes. The bioincompatibility of CU seems to be responsible for the greater amount of cAMP in adherent cells, and this parameter could provide an index for assessing biocompatibility.

Prostacyclin secretion by human endothelial cells grown on carbodiimide cross-linked proteins: an assessment of the cytocompatibility of a substratum.

Prostacyclin production by human umbilical vein endothelial cells cultured on carbodiimide cross-linked albumin and/or gelatin was quantified during the exponential growth phase and in confluent cultures as a response to arachidonic acid stimulus. In confluent cultures, basal production of prostacyclin measured by radioimmunoassay of the stable metabolite 6-keto-PGF1 alpha was comparable for both substrates to a control culture. Maximal release of prostacyclin occurred during the first 24 h following cell seeding and these values were significantly higher in media from cultures performed on membranes. In both cases, PGI2 production decreased as cell density increased. After stimulation with 20 microM arachidonic acid for 20 min, media from confluent cells grown on membranes contained slightly greater amounts of PGI2 than control culture medium. These results indicate involvement of substratum in PGI2 Release. Early enhancement of PGI2 secretion could improve biocompatibility of membranes by preventing platelet aggregation.

Non-saponifiable fraction of cocoa shell butter: effect on rat and human skin fibroblasts.

Synopsis Non-saponifiable lipid fraction (ICSB) extracted from cocoa shell butter was solubilized in dimethylformamide (DMF) and analysed for its biological activity on growth of rat and human fibroblasts. Non-saponifiables (10 mug ml(-1)) partially protected cells from toxicity of DMF (1%) and allowed the growth of fibroblasts cultivated in optimal conditions (10% fetal calf serum-FCS, 37 degrees C) or improved the survival of cells maintained in altered conditions (2.5% FCS, 35 degrees C). At higher concentration (ICSB 50 mug ml(-1), DMF 1%), the protective effect was suppressed. ICSB was fractionated by chromatography into four compounds: sterols, terpenic alcohols, tocopherols and hydrocarbons +/- carotenoids. We found that biological activity of ICSB was mostly due to the major fraction containing sterols.

IL-1-induced procoagulant and fibrinolytic activities of human endothelium grown on carbodiimide crosslinked proteins.

We examined the regulation of procoagulant activity and the production of fibrinolytic components by human vascular endothelium grown on coating membranes of gelatin, pure or mixed with albumin, crosslinked by carbodiimide ((G)C, (AG)C) in comparison with plastic culture dishes. Confluent monolayers were stimulated by human recombinant interleukin (IL-1 beta) and responses in terms of tissue factors like procoagulant activity, tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) were followed for up to 72 h. Procoagulant activity of cell extracts displayed similar patterns whatever the substratum tested. Quantitative immunological assays revealed a 2-fold increase in tPA antigen released from monolayers grown on (G)C and on (AG)C compared to cells grown on plastic. Exposure of monolayers to IL-1 beta reduced the secretion of tPA antigen which still reached higher values on coated than on uncoated substratum. We found that the quasi-totality of tPA formed stable complexes with PAI-1, thereby suppressing measurable fibrinolytic activity. IL-1 beta stimulated the release of PAI-1 antigen quantified by immunoassay and the kinetics of secretion were comparable on both coated and uncoated substratum.

Comparative behaviour of L-929 fibroblastic and human endothelial cells onto crosslinked protein substrates.

Studies were carried out to compare the behaviour of human umbilical vein endothelial cells (HUVEC) and L-929 fibroblastic cells towards proteins crosslinked by glutaraldehyde (GTA) or carbodiimide (CDI) proposed for coating of vascular prostheses. CDI crosslinking of bovine serum albumin used alone, or mixed with gelatin, allowed higher rates of cell growth and DNA synthesis than GTA crosslinking independent of cells. Assessment of the plating efficiency revealed a similar behaviour of both cells towards membranes and reference plastic surface in terms of percentages of bound cells. HUVEC proliferation onto CDI crosslinked gelatin and/or albumin membranes did not differ significantly whereas the growth of L-929 was enhanced onto gelatin albumin membranes in comparison with both gelatin membranes and the reference surface. The analysis of DNA synthesis corroborated the results of the growth curves and elicited a delay of the growth phases in HUVEC cultured onto CDI crosslinked membranes, unlike the L-929 fibroblast.

F.p.l.c. analysis of the leakage products of proteins crosslinked on Dacron vascular prostheses.

The haemocompatibility of vascular, Dacron prostheses was improved by coating with albumin and/or collagen crosslinked with glutaraldehyde (GTA) or carbodiimide (CDI). F.p.l.c. (fast protein liquid chromatography) analysis of the products desorbed from polymeric matrices incubated in physiological conditions for periods extended up to 10 d did not detect the monomers or polymers of collagen and albumin but small amounts of degradation products, the molecular weights of which were less than 45,000, thus minimizing an eventual immunogenic response after implantation. However GTA and CDI matrices required extensive washing to neutralize the cytotoxic effect of GTA and achieve the release of CDI from protein complexes.