Conformational changes of plasma fibronectin detected upon adsorption to solid substrates: a spin-label study

Changes in local environment of the free sulfhydryl groups in plasma fibronectin upon adsorption of the protein to polystyrene beads have been examined by electron spin resonance (ESR) spin-label spectroscopy. The two free sulfhydryl groups per subunit of plasma fibronectin were modified chemically with an [15N, 2H]maleimide spin-label. For soluble fibronectin, both free sulfhydryl groups are shown to be in confined environments as evidenced from the labeled protein exhibiting a strongly immobilized ESR spectrum as described previously using [14N, 1H]maleimide spin-labels [Lai, C.-S., & Tooney, N. M. (1984) Arch. Biochem. Biophys. 228, 465-473]. When the labeled protein was adsorbed to the beads, half of the strongly immobilized component was found to convert into a weakly immobilized component, a result indicating that one of the two labeled sites becomes exposed and exhibit a fast tumbling motion. Experiments conducted using various spin-labeled fibronectin fragments suggest that the newly exposed labeled site is located between the DNA-binding and the cell-binding regions of the molecule. The data obtained indicate that, upon adsorption to polystyrene beads, plasma fibronectin undergoes a conformational change through which the buried free sulfhydryl group near the cell-binding region of the molecule is exposed. This observation may have important implications regarding the expression of cell adhesive properties of the fibronectin molecule.     

Adhesive substrates for fibronectin

In order to promote cell attachment, fibronectin must first undergo activation by a suitable substrate. In this study, 52 materials have been surveyed for their ability (a) to bind fibronectin, (b) to activate the cell-adhesive property of fibronectin, and (c) to support the growth of cells. Many plastics, polysaccharides, metals, and ceramics were found to support cell growth as well as the fibronectin-dependent attachment of cells. Several other substrates have been identified that were inactive in promoting either cell attachment or growth. Hydrophobic substrates were found to be active in fibronectin activation, whereas hydrophilic substrates were found to be inactive. Since fibronectin binds to substrata of extremely varied chemical composition, it is clear that the binding of fibronectin to such substrata is nonspecific in nature. Since protein pretreatment of all substrata, except collagen and poly(L-lysine), abolished the physical binding of fibronectin, the binding of fibronectin to artificial substrata is probably ascribable to a nonspecific hydrophobic protein-substratum interaction. In contrast, several lines of evidence indicate that the interaction between fibronectin and collagen displays biological specificity. Poly(hydroxyethylmethacrylate)(poly(HEMA)), which has previously been shown to be nonadhesive for cells, is demonstrated here to be unique in its inability to bind fibronectin. Addition of one part per million of an adhesive polymer to poly(HEMA) permits fibronectin binding to occur.     

Surface functionalization and dynamics of polymeric cell culture substrates

1. Download : Download high-res image (160KB)
2. Download : Download full-size image

     

Surface creasing instability of soft polyacrylamide cell culture substrates

Efforts to understand and engineer cell behavior in mechanically soft environments frequently employ two-dimensional cell culture substrates consisting of thin hydrogel layers with low elastic modulus supported on rigid substrates to facilitate culturing, imaging, and analysis. Here we characterize how an elastic creasing instability of the gel surface may occur for the most widely used soft cell culture substrate, polyacrylamide hydrogels, and show that stem cells respond to and change their behavior due to these surface features. The regions of stability and corresponding achievable ranges of modulus are elucidated in terms of the monomer and cross-linker concentrations, providing guidance for the synthesis of both smooth and creased soft cell substrates for basic and applied cell engineering efforts.     

Pericellular substrates of human mast cell tryptase: 72,000 dalton gelatinase and fibronectin.

Migrating cells degrade pericellular matrices and basement membranes. For these purposes cells produce a number of proteolytic enzymes. Mast cells produce two major proteinases, chymase and tryptase, whose physiological functions are poorly known. In the present study we have analyzed the ability of purified human mast cell tryptase to digest pericellular matrices of human fibroblasts. Isolated matrices of human fibroblasts and fibroblast conditioned medium were treated with tryptase, and alterations in the radiolabeled polypeptides were observed in autoradiograms of sodium dodecyl sulphate polyacrylamide gels. It was found that an M(r) 72,000 protein was digested to an M(r) 62,000 form by human mast cell tryptase while the plasminogen activator inhibitor, PAI-1, was not affected. Cleavage of the M(r) 72,000 protein could be partially inhibited by known inhibitors of tryptase but not by aprotinin, soybean trypsin inhibitor, or EDTA. Fibroblastic cells secreted the M(r) 72,000 protein into their medium and it bound to gelatin as shown by analysis of the medium by affinity chromatography over gelatin-Sepharose. The soluble form of the M(r) 72,000 protein was also susceptible to cleavage by tryptase. Analysis using gelatin containing polyacrylamide gels showed that both the intact M(r) 72,000 and the M(r) 62,000 degraded form of the protein possess gelatinolytic activity after activation by sodium dodecyl sulphate. Immunoblotting analysis of the matrices revealed the cleavage of an immunoreactive protein of M(r) 72,000 indicating that the protein is related to type IV collagenase. Further analysis of the pericellular matrices indicated that the protease sensitive extracellular matrix protein fibronectin was removed from the matrix by tryptase in a dose-dependent manner. Fibronectin was also susceptible to proteolytic degradation by tryptase. The data suggest a role for mast cell tryptase in the degradation of pericellular matrices.     

Sponge cell culture: a comparative evaluation of adhesion to a native tissue extract and other culture substrates

Hexactinellids are deep water sponges that possess syncytial rather than cellular tissues. In order to investigate the syncytial character of the tissue of these unusual sponges, primary cultures were developed using a substrate of acellular tissue extract (ATE) that promotes the adhesion and spreading of sponge tissues. Primary cultures of the hexactinellid sponge Rhabdocalyptus dawsoni, grown on this substrate, form thinly spread, multinucleate, confluent tissue masses which exhibit active cytoplasmic streaming. Sponge tissue adhered equally well to commercial substrates of concanavalin A and poly-l-lysine, but did not adhere to chicken collagen. Acellular tissue extracts prepared from demosponges, which are known to be cellular, also promoted adhesion and spreading of cells from those sponges. Scanning electron microscopy showed adherent Rhabdocalyptus tissue to have an uninterrupted, smooth membrane covering the entire culture, unlike primary cultures of the cellular demosponge, Haliclona sp., which consisted of numerous individual cells. Tissue from freshly collected sponges adhered preferentially to ATE from a conspecific. However, after continued wounding, tissue adhered indescriminately to any substrate. The tissue extract congealed if added to sea water or 10 mM CaCl(2), forming a white, cloudy solid, which could be fixed and sectioned for transmission electron microscopy. Thin sections of the congealed extract showed it to contain membranes but no visible collagen fibrils.     

Fibroblast cell behavior on bound and adsorbed fibronectin onto hyaluronan and sulfated hyaluronan substrates

The effect of fibronectin protein (Fn) coating onto polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) on fibroblast cell adhesion was analyzed. The Hyal or HyalS were coated and grafted on the glass substrate by a photolithographic method. The Fn coating was achieved by two different routes: the immobilization of Fn by covalent bond to the polysaccharide layers and the simple adsorption of Fn onto Hyal and HyalS surfaces. AFM, SEM, and ATR-FTIR techniques were used for the chemical and topographical characterization of the surfaces. According to AFM and SEM data, the surface topography was dependent on the method used to cover the polysaccharide layers with the protein. ATR-FTIR analysis supplied information about the rearrangement of Fn after the interaction (adsorption or binding) with the Hyal and the HyalS. The conformational changes of the Fn were minimal when it was simply adsorbed on HyalS surfaces and larger once bound, whereas on the Hyal layer the protein underwent a bigger conformational change once adsorbed and covalently grafted. Then, the biological characterization was carried out by analyzing the human diploid skin fibroblasts adhesion on these surfaces. The morphology of fibroblasts was evaluated by SEM, whereas the dynamics of fibroblasts movement were recorded by a time-lapse system. Cell variations in area, perimeter, and length were analyzed at 2, 4, and 6 h. It was found that the addition of Fn (covalently bound or merely adsorbed) was fundamental in the promotion of fibroblasts adhesion and spreading. The greatest adhesion occurred onto HyalS layers covered by the adsorbed Fn.     

Modulation of cell attachment to culture support by pH, fibronectin, hemin, and cobalt protoporphyrin

When chick embryo fibroblasts were seeded in the presence of minimum essential medium supplemented with 1% (v/v) horse serum, the rate of cell attachment, after 1 hr incubation, was less than 5% at pH 6.5 and about 50% and 80% at pH 7.5 and pH 8.3, respectively. If, however, cultures were pretreated with fibronectin, a substance that promotes cell adhesion, a high rate of cell attachment was also observed at pH 6.5. Two other compounds of totally different chemical nature, cobalt-protoporphyrin (CoPP) and hemin, also enhanced cell attachment at pH 6.5. CoPP was shown to increase the synthesis of proteins, but it did not affect the intracellular heme content of cells incubated at pH 6.5. The possibility that CoPP, and presumably also hemin, induce cell attachment by promoting the synthesis of a fibronectin like protein is discussed.     

Effects of various substrates on human hepatoblastoma and hepatoma cell culture

The effects on human hepatoblastoma (HuH-6) and hepatoma (HuH-7) cell lines of collagen type I (CI), type IV (CIV), fibronectin (FN) and laminin (LAM) were investigated. CI and CIV promoted almost equally the attachment of both cell lines more strikingly than did FN or LAM. CI and CIV were also superior to FN or LAM in supporting the growth of HuH-6. These substrates, however, had no appreciable effect on the growth of HuH-7. The amount of alpha-fetoprotein (AFP) and albumin secreted in HuH-6 was found to be higher on FN- and LAM- coated substrates than on the other matrix material- coated ones. HuH-7 exhibited increased levels of AFP on LAM- coated substrates 4 days after plating. These results indicate that the ability of extracellular matrix materials to enhance attachment and/or growth is different from that to enhance AFP and albumin production in HuH-6 and probably in HuH-7.     

Concurrent modulation of cell surface fibronectin and adhesion to fibronectin in hepatoma cells

Rat hepatoma cells grown in vitro were poorly adhesive to plastic surfaces coated with fibronectin and lacked cell surface fibronectin matrix. They synthesized soluble fibronectin into the medium. The cell surface fibronectin matrix and the ability to attach to fibronectin-coated surface were restored in the 7777 cells upon passage as a tumor in rats and by coculturing these cells with normal liver-derived cells in vitro. Fibronectin matrix and the ability of cells to attach to fibronectin were thus modulated in a coordinated fashion, suggesting that the formation of a cell surface fibronectin matrix is dependent on the cell surface property that enables cells to interact with fibronectin.     

Quantification of streptavidin adsorption in microtitration wells

Streptavidin-coated microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantified by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm(2) (370 ng) and consequently the theoretical adsorption capacity of a C12-format microtitration well (200 microl liquid, coated area 1.54 cm(2)) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350-380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive fluorescence and protein measurement methods in the microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.     

Quantification of tissue fibronectin from terminal villi of placenta.

Tissue fibronectin (TFn) was solubilized from the terminal villi of perfused human placentas by sequential chemical extractions and plasmin digestion. Alternatively, plasmin digestion of intact tissue solubilized all the TFn, which amounted to 1.8-2.9% of the dry weight of the villi. Concomitantly, 69% of the tissue was solubilized. The non-equilibrium competitive e.l.i.s.a. (enzyme-linked immunoabsorbent assay), in which the TFn was immunologically identical with plasma fibronectin (PFn), was used for the quantification of TFn. This study demonstrates that the bulk of TFn can be obtained in a form that can be quantified by e.l.i.s.a. and that TFn is immunologically identical with PFn. Thus the fibronectin molecule is not significantly altered as it is incorporated into the connective-tissue matrix and could exchange with PFn.     

Quantification of l-alanyl-l-glutamine in mammalian cell culture broth: Evaluation of different detectors

l-Alanyl-l-glutamine (also known as Ala-Gln or GlutaMAX) is widely used as a stable l-glutamine source in cell culture for the production of biopharmaceuticals. System approaches for the optimization of production processes require the analysis of all major substrates and products. We have compared four alternative detection systems for l-alanyl-l-glutamine in culture broth. Matrix effects prevented the use of ultraviolet or evaporative light scattering detection. Fluorescence detection used in routine amino acid protocols is compatible with culture broth and has a broad linear dynamic range. Mass spectrometry has superior sensitivity and can be integrated into quantitative metabolomic workflows.     

Quantification of chemical–polymer surface interactions in microfluidic cell culture devices

Microfluidic cell culture devices have been used for drug development, chemical analysis, and environmental pollutant detection. Because of the decreased fluid volume and increased surface area to volume ratio, interactions between device surfaces and the fluid is a key element that affects the performance and detection accuracy of microfluidic devices, particularly if fluid is recirculated by a peristaltic pump. However, this issue has not been studied in detail in a microfluidic cell culture environment. In this study, chemical loss and contaminant leakage from various polymer surfaces in a microfluidic setup were characterized. The effects of hydrophilic coating with Poly (vinyl alcohol), Pluronic® F‐68, and multi‐layer ionic coating were measured. We observed significant surface adsorption of estradiol, doxorubicin, and verapamil with PharMed® BPT tubing, whereas PTFE/BPT and stainless steel/BPT hybrid tubing caused less chemical loss in proportion to the fraction of BPT tubing in the hybrid system. Contaminants leaching out of the BPT tubing were found to be estrogen receptor agonists as determined by estrogen‐induced green fluorescence expression in an estrogen responsive Ishikawa cell line and also caused interference with an estradiol enzyme‐linked immunosorbent assay (ELISA) assay. Stainless steel/BPT hybrid tubing caused the least interference with ELISA. In summary, polymer surface and chemical interactions inside microfluidic systems should not be neglected and require careful investigations when results from a microfluidic system are compared with results from a macroscale cell culture setup. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Quantification of cell culture factors affecting recombinant protein yields in baculovirus-infected insect cells

An experimental study was undertaken to quantify the effects of infection cell density, medium condition, and surface aeration on recombinant protein yields in insect cells. In the absence of surface aeration and fresh medium, insect cells generated higher product yields (on a per cell basis) when infected with recombinant baculovirus at low cell densities, LCD (3 x 10(5)-4 x 10(5) cells/mL), than at high cell densities, HCD (>0.9 x 10(6) cells/mL), for two distinct baculovirus types. Surface aeration of a HCD culture infected in spent medium improved beta-glactosidase yields 5-fold over the nonaerated case. Surface aeration and medium replenishment improved beta-galactosidase yields of a HCD culture by 20-fold (compared to a 1.6-fold improvement for a LCD culture), resulting in cultures with productivties that were independent of the cell density at infection.     

Three-dimensional culture regulates Raf-1 expression to modulate fibronectin matrix assembly

Oncogenic transformation has been associated with decreased fibronectin (FN) matrix assembly. For example, both the HT-1080 fibrosarcoma and MAT-LyLu cell lines fail to assemble a FN matrix when grown in monolayer culture (2-dimensional [2D] system). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3-dimensional [3D] system). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression compared with cells grown in monolayer culture. This effect is associated with reduced Raf-1 mRNA levels as determined by quantitative RT-PCR and not proteasome-mediated degradation of endogenous Raf-1. Interestingly, transient expression of a Raf-1 promoter-reporter construct demonstrates increased Raf-1 promoter activity in 3D, suggesting that the transition to 3D culture may modulate Raf-1 mRNA stability. Finally, to confirm that decreased Raf-1 expression results in increased FN matrix assembly, we used both pharmacological and small interfering RNA knockdown of Raf-1. This restored the ability of cells in 2D culture to assemble a FN matrix. Moreover, overexpression of Raf-1 prevented FN matrix assembly by cells cultured in 3D, resulting in decreased aggregate compaction. This work provides new insight into how the cell microenvironment may influence Raf-1 expression to modulate cell-FN interactions in 3D.     

Properties and fate of plasma fibronectin bound to the tissue culture substratum

Plasma fibronectin (pFn) is a serum protein which, when adsorbed to a glass or plastic substratum, mediates the adhesion of fibroblasts in culture. We have studied some of the details of its adsorption and subsequent fate. By using ¹²⁵l-labeled pFn, we show that a substratum incubated with pFn adsorbs approximately 0.4 μg/cm² pFn (a monomolecular layer), and one incubated with medium containing serum adsorbs approximately 7 ng/cm² pFn (a 12-fold enrichment relative to a random selection of the soluble proteins). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) suggests the bound serum proteins (eluted with SDS) are primarily BSA and β-globulins. The bound pFn adheres so tightly, though, that most resists elution, as assayed (1) with pFn radioiodinated before binding, (2) with pFn radioiodinated after binding, or (3) by the cell spreading activity of the bound pFn retained after SDS treatment. Under culture conditions, there is a continuous “turnover” of substratum-bound pFn: soluble pFn can bind to a serum-coated substratum, while bound pFn is gradually removed by incubation with serum proteins. The presence of fibroblasts increases the rate of this removal several-fold. By SDS-PAGE the material removed (as well as that eluted from the substratum with SDS after cell detachment) is intact pFn or large (possibly proteolytically generated) fragments. Thus, pFn binds preferentially to the tissue culture substratum, but can be removed subsequently by the combined action of cells and other serum proteins.     

A strategy for high cell density culture of heterotrophic microalgae with inhibitory substrates

Substrate inhibition is one of the major problems preventing high cell densities of microalgae in heterotrophic culture, so the possibility of overcoming the problem by various culture techniques was examined. It was found that perfusion culture may be the most appropriate technique for high cell densities in heterotrophic culture using inhibitory substrates. An experimental example in which a hollow fibre cell recycle system (HFCRS) was employed to achieve high cell densities of Chlamydomonas reinhardtii on acetate under heterotrophic conditions of growth was demonstrated. The cell density in the HFCRS was much higher than that reported in the literature for this species.