Surface-attached Cells Research Articles

Bio Imaging of Intracellular NO Production in Single Bone Cells After Mechanical Stimulation

We show the intracellular upregulation of NO production after mechanical stimulation, an essential chemical signal in bone remodeling. This is done in real time using the fluorescent chromophore DAR-4M AM. Differences in cellular response to mechanical stimulation of different regions of a single cell were observed. Osteocytes are the most abundant bone cells that are believed to be the mechanosensors of bone, responding to mechanical stresses in interstitial fluid flow through the canaliculi. Under mechanical load, chemical signals such as NO play a key role in the activity of osteoblasts/osteoclasts that regulate bone remodeling. Despite the importance of NO in signaling, its real-time detection has proved challenging. This is largely because of the short NO half-life (typically approximately 0.1-5 s). Here, we show the upregulation of intracellular NO production in single osteocytes under localized mechanical stimulation. We used the chromophore DAR-4M AM for NO detection. This is loaded into surface-attached MLO-Y4 osteocyte-like and MC3T3-E1 osteoblast-like cells that are subjected to a localized mechanical stimulation using optical tweezers or a microneedle tip. DAR-4M AM is membrane-permeable and chelates NO, forming a stable, fluorescent compound, which is visible with a rhodamine filter. Nonstimulated MLO-Y4 and MC3T3-E1 cells showed basal NO production levels, as indicated by a gradual increase in their fluorescence intensity. Localized mechanical stimulation of single MC3T3-E1 cells and MLO-Y4 cells by optical tweezers (150-550 pN, 0.5-3 Hz, 1 minute) showed a nearly 15-30% increase, whereas MLO-Y4 cells stimulated by a microneedle (10-20 nN, 1 minute) showed nearly 15-16% increase relative to their nonstimulated state. Furthermore, stimulation of a single cell process by a microneedle resulted in a 2-10% increase in the fluorescence intensity. NO is essential for mechanically induced bone remodeling and is a meaningful parameter for measuring bone cell activation after mechanical loading. Here we show NO upregulation in individual bone cells after a localized mechanical stimulation. We also show that both the cell body and the cell processes might be involved in mechanosensing. This technique allows characterization of the mechanosensitivity of different parts of a single osteocyte. This opens up the possibility to uncover the complexities and function of single osteocytes in the dynamic process of bone remodeling.

Polysaccharide differences between planktonic and biofilm-associated EPS from Pseudomonas fluorescens B52

The polysaccharides associated with free (planktonic) and surface-attached (biofilm) cells from cultures of Pseudomonas fluorescens strain B52 were compared. Variations in the attached matrix due to surface material (glass or stainless steel) were also analyzed. Two digestion methods were used to optimize the recoveries of sugars, uronic acids and acidic substituents. The yield of analyzable material after digestion reached 90% for the material associated to the biofilms, though only 20–30% for that bound to planktonic cells. The polysaccharide(s) in the biofilm had glucuronic and guluronic acids as main components, besides rhamnose, glucose and glucosamine. The proportion of glucuronic to guluronic acid was higher in the polysaccharide(s) found in biofilms formed on stainless steel than in those on glass.

Collagen/Poloxamine Hydrogels: Cytocompatibility of Embedded HepG2 Cells and Surface-Attached Endothelial Cells

The effects of cross-linked poloxamine hydrogels on the cellular function of embedded HepG2 cells and surface-attached endothelial cells were assessed. HepG2 cells embedded within collagen/poloxamine-methacrylate gel survived photo-cross-linking (MTT viability, 78%). There was a gradual increase in cell number during the first week. The cumulative secretion of alpha1-antitrypsin by HepG2 cells showed an almost linear profile. However, lower levels for the collagen/poloxamine-methacrylate matrix were observed when compared with collagen. Endothelial cells attached poorly to poloxamine gels without collagen (alamarBlue reduction ranged from 36 to 63%) and did not spread well. The addition of collagen led to spread cells and alamarBlue reduction levels of 75-93% (24 h after seeding). On day 5, some detachment was noted through analysis of vascular endothelial cadherin staining. Finally, the collagen-containing matrix was used to prepare cylindrical modules containing HepG2 cells to show the utility of this material in modular tissue constructs. A fluorescent cytoplasmic tracer, Vybrant CFDA SE, showed that embedded cells remained viable for more than 2 months, confirming the good cytocompatibility of collagen/poloxamine-methacrylate in the form of modules. The suitability of these modules for preparing uniform, scaleable, and vascularized constructs remains to be demonstrated.

Biofilm formation of Pseudomonas putida IsoF: the role of quorum sensing as assessed by proteomics

Pseudomonas putida strains are frequently isolated from the rhizosphere of plants and many strains promote plant-growth, exhibit antagonistic activities against plant pathogens and have the capacity to degrade pollutants. Factors that appear to contribute to the rhizosphere fitness are the ability of the organism to form biofilms and the utilization of cell-to-cell-communication systems (quorum sensing, QS) to co-ordinate the expression of certain phenotypes in a cell density dependent manner. Recently, the ppu QS locus of the tomato rhizosphere isolate P. putida Iso F was characterized and an isogenic QS-negative ppuI mutant P. putida F117 was generated. In the present study we investigated the impact of QS and biofilm formation on the protein profile of surface-associated proteins of P. putida IsoF. This was accomplished by comparative proteome analyses of the P. putida wild type IsoF and the QS-deficient mutant F117 grown either in planktonic cultures or in 60 h old mature biofilms. Differentially expressed proteins were identified by peptide mass fingerprinting and database search in the completed P. putida KT2440 genome sequence. The sessile life style affected 129 out of 496 surface proteins, suggesting that a significant fraction of the bacterial genome is involved in biofilm physiology. In surface-attached cells 53 out of 484 protein spots were controlled by the QS system, emphasizing its importance as global regulator of gene expression in P. putida IsoF. Most interestingly, the impact of QS was dependent on whether cells were grown on a surface or in suspension; about 50% of the QS-controlled proteins identified in planktonic cultures were found to be oppositely regulated when the cells were grown as biofilms. Fifty-seven percent of all identified surface-controlled proteins were also regulated by the ppu QS system. In conclusion, our data provide strong evidence that the set of QS-regulated proteins overlaps substantially with the set of proteins differentially expressed in sessile cells.

Identification of AbrB-regulated genes involved in biofilm formation by Bacillus subtilis.

Bacillus subtilis is a ubiquitous soil bacterium that forms biofilms in a process that is negatively controlled by the transcription factor AbrB. To identify the AbrB-regulated genes required for biofilm formation by B. subtilis, genome-wide expression profiling studies of biofilms formed by spo0A abrB and sigH abrB mutant strains were performed. These data, in concert with previously published DNA microarray analysis of spo0A and sigH mutant strains, led to the identification of 39 operons that appear to be repressed by AbrB. Eight of these operons had previously been shown to be repressed by AbrB, and we confirmed AbrB repression for a further six operons by reverse transcription-PCR. The AbrB-repressed genes identified in this study are involved in processes known to be regulated by AbrB, such as extracellular degradative enzyme production and amino acid metabolism, and processes not previously known to be regulated by AbrB, such as membrane bioenergetics and cell wall functions. To determine whether any of these AbrB-regulated genes had a role in biofilm formation, we tested 23 mutants, each with a disruption in a different AbrB-regulated operon, for the ability to form biofilms. Two mutants had a greater than twofold defect in biofilm formation. A yoaW mutant exhibited a biofilm structure with reduced depth, and a sipW mutant exhibited only surface-attached cells and did not form a mature biofilm. YoaW is a putative secreted protein, and SipW is a signal peptidase. This is the first evidence that secreted proteins have a role in biofilm formation by Bacillus subtilis.

Iron corrosion by novel anaerobic microorganisms.

Corrosion of iron presents a serious economic problem. Whereas aerobic corrosion is a chemical process, anaerobic corrosion is frequently linked to the activity of sulphate-reducing bacteria (SRB). SRB are supposed to act upon iron primarily by produced hydrogen sulphide as a corrosive agent and by consumption of 'cathodic hydrogen' formed on iron in contact with water. Among SRB, Desulfovibrio species--with their capacity to consume hydrogen effectively--are conventionally regarded as the main culprits of anaerobic corrosion; however, the underlying mechanisms are complex and insufficiently understood. Here we describe novel marine, corrosive types of SRB obtained via an isolation approach with metallic iron as the only electron donor. In particular, a Desulfobacterium-like isolate reduced sulphate with metallic iron much faster than conventional hydrogen-scavenging Desulfovibrio species, suggesting that the novel surface-attached cell type obtained electrons from metallic iron in a more direct manner than via free hydrogen. Similarly, a newly isolated Methanobacterium-like archaeon produced methane with iron faster than do known hydrogen-using methanogens, again suggesting a more direct access to electrons from iron than via hydrogen consumption.

Colored moderately thermophilic bacteria in paper-machine biofilms.

Biofilms cause several problems in papermaking. This report describes a microbiological survey of colored biofilms in six paper and board machines, including two case studies of outbreaks of colored slimes in which the causative bacteria were found. A total of 95 pink-, red-, orange- or yellow-pigmented strains were isolated. Nearly all (99%) of the strains grew at 52 degrees C, 72% grew at 56 degrees C, but only 30% grew at 28 degrees C, indicating that most of the strains were moderately thermophilic. Biofilm formation potential and biocide susceptibility of the strains were analyzed with a microtiter plate assay. In the presence of 5 ppm of methylene bisthiocyanate or 2,2-dibromo-3-nitrilopropionamide in paper-machine water, 55 strains formed biofims. Moreover, 39 strains increased biofilm production by 5-753% in the presence of biocide, suggesting that biocide concentrations inhibitory to planktonic but not to surface-attached cells may actually promote biofouling. The cells may have inactivated a portion of the biocides, as the cell density in this assay was high, corresponding to the highest cell densities occurring in the circulating waters. Four groups of colored bacteria that were isolated from several mills were identified. Pink-pigmented Deinococcus geothermalis and red-pigmented Meiothermus silvanus occurred as common primary biofilm-formers in paper machines. This report is the first description of the involvement of Meiothermus species in red-slime formation in the paper industry. The third group of bacteria (putative new species related to Roseomonas) contained strains that were not biofilm formers, but which were commonly found in slimes of neutral or alkaline machines. The fourth group contained red-pigmented biofilm-forming strains representing a novel genus of alpha- Proteobacteria related to Rhodobacter. Many colored paper-machine bacteria are species previously known from microbial mats of hot springs. Some characteristics of the bacterial groups are described here in order to facilitate their recognition in future cases of colored-slime outbreaks in the paper industry.

Highly sensitive ELISA-based assay for quantifying protein levels in neuronal cultures

A simple assay for quantifying the expression level of proteins in situ is presented. The assay is performed directly in culture plates and simply requires incubating the cells (either fixed or in live cultures) with the appropriate antibody against the desired protein. Cells are then incubated with a peroxidase-conjugated secondary antibody followed by incubation with the soluble peroxidase substrate—tetramethylbenzidine. Antibody binding levels are quantified in an ELISA-based protocol by reading optical density at 450 nm. The assay is applicable for any surface-attached cell culture. The assay is highly sensitive, allowing detection of expressed proteins in a monolayer of cell cultured in a 96 well plate. The assay was optimized for a neuronal cell-line—P19. These are embryonal carcinoma cells which differentiate into neurons after treatment with retinoic acid. The neurons do not survive well in 96 well plates and are thus plated in 24 or 48 well plates. We demonstrate the use of the method for an in vivo labeling of neurons by quantifying epitopes which are exposed upon nerve stimulation. Advantages of a routine ELISA protocol in quantification over wide range of sensitivity and in performing simultaneous large number of repetitions and different controls are thus fully extended to live cell cultures. Themes: Cell biology Topics: Staining, tracing, and imaging techniques

N‐acetylglucosamine and adenosine derivatized surfaces for cell culture: 3T3 fibroblast and chicken hepatocyte response

3T3 fibroblasts and primary chicken hepatocytes were cultured on derivatized polystyrene surfaces to examine the effect of cell-specific ligands on cellular morphology and growth. Surfaces were prepared by derivatizing chloromethylated polystyrene with N-acetylglucosamine (GlcNAc; recognized by the chicken asialoglycoprotein receptor) and adenosine (not recognized by adult hepatocytes). These surfaces were compared with tissue culture polystyrene (TCPS), acid-cleaned glass, and the unmodified chloromethylated polystyrene. The spreading, cytoskeletal structure and growth of the fibroblasts following attachment to these surfaces were examined. The extent of attachment, total protein levels, and DNA contents for surfaces-attached chicken hepatocytes were also measured. Fibroblast spreading was greatest on polymer surfaces derivatized with GlcNAc, whereas cytoskeletal structure and growth rate were independent of surface chemistry. Although chicken hepatocytes attached most efficiently to the GlcNAc derivatized polymer, the total protein and DNA levels of the surface-attached cells were not affected. In anticipation of the application of these polymers for cell culture and hybrid artificial organ design, the GlcNAc-derivatized polystryrene was fabricated into porous microcarriers. Fibroblasts grew avidly on the microcarriers, whereas chicken hepactocytes adhered well to the formed large aggregates around the microcarriers.

Biofilms and the technological implications of microbial cell adhesion

Biofilms are a collection of cells entrapped within a gelatinous matrix comprising mostly insoluble extracellular polymers that the cells secrete. Although the term is applied mostly to bacterial cells and their secreted insoluble exopolymers, any biologically active layer of cells (microbial, plant, or mammalian cells) can be considered a biofilm. Any surface in contact with a biological fluid is a potential target surface for microbial cell adhesion. Once cells are attached, subsequent growth and replication of surface-attached cells, cell exopolymer production, further recruitment of planktonic cells from the fluid phase, and various biofilm detachment processes constitute what is collectively known as biofilm formation and persistence. Biofilms can play both beneficial or detrimental roles depending on whether their formation within a specific system is intentional or inadvertent. This article will review both the current and emerging technological implications of bacterial cell adhesion and biofilm formation including biomaterials preparation to prevent bacterial infections of medical implants; development of novel antibiotic therapies to control biofilm-bound bacteria; designer nanocrystalline filaments called “bionites”, fabricated from strands of bacteria, that possess unusual magnetic, optical and biocatalytic properties; specific hazardous waste detoxification by immobilized recombinant bacteria; improved recombinant plasmid retention within biofilm populations; and stable biosensors.

Screening of whole-cell immobilization procedures for the Δ1-dehydrogenation of steroids in organic medium

Different whole-cell immobilization methods and conditions were tested for the Δ 1-dehydrogenation of 6-α-methyl-hydrocortisone-21-acetate with Arthrobacter simplex cells, in n-decane-1-ol and chloroform. Among the entrapment methods, polyurethane foams gave the best productivity of n-decane-1-ol, but could not be used in chloroform. Partition and diffusional barriers appeared to be the factors limiting productivity in entrapped-cell systems. The coentrapment of enzyme-stabilizing (glycerol, sucrose, sodium sulfate, monosodium glutamate) or hydrophobic additives did not significantly improve dehydrogenation rates in chloroform, although in some cases higher activities resulted in n-decane-1-ol. Dehydration of κ-carrageenan-immobilized cells lowered the productivity in both solvents. The use of cell adsorption methods on silica-based carriers produced biocatalysts that in some cases were more active in chloroform than the entrapped cells. However, the surface-attached cells appeared to be more sensitive to solvent toxicity, more hydrophilic supports generally giving higher dehydrogenation rates. The estimated partition coefficients for the substrate between several of the tested supports and the two solvents are also presented. High partition ratios followed high measured activities in entrapment matrices but not in Celite (cell adsorption matrix). Activities in chloroform were always very low (less than 1 μmol product per hour and gram of cell dry weight). Polyurethane entrapment with n-decane-1-ol as a reaction medium was the most promising system, with measured dehydrogenation rates up to 40 times higher.

Fibroblast and hepatocyte behavior on synthetic polymer surfaces

Biodegradable poly(phosphoesters) with varying side group chemistry and copolymers of styrene and methyl vinyl ketone (MVK) with varying degrees of hydrophobicity were used to study the growth and behavior of surface-attached fibroblasts and hepatocytes. Mouse 3T3 fibroblasts and chicken embryo fibroblasts attached and proliferated on all of the polymers tested. Fewer cells attached to copolymers of styrene and MVK than to glass or tissue culture polystyrene controls; cell attachment to several poly(phosphoester) surfaces was indistinguishable from controls. The mean speed of fibroblast migration was faster on surfaces where fewer cells attached (59 to 84 microns/h on low attachment surfaces compared with 40 to 46 microns/h on high attachment surfaces). When surface-attached cells were stained with fluorescently labeled phalloidin, only a fraction of the cells on low attachment surfaces were shown to have prominent arrays of actin filament bundles. Chicken hepatocytes also attached to the polymer surfaces. When a suspension containing a large number of cells was placed over the polymer surfaces, approximately 50% of the hepatocytes attached during the first 9 h. Surprisingly, hepatocyte attachment and viability in culture were relatively insensitive to the chemistry of the synthetic polymer substrates. Cell number increased by about a factor of 2 over the first 48 h of culture, then decreased back to approximately 50% of initial cell number over the next several days. Cell morphology did depend on the chemical structure of the substrates.

7,12-Dimethylbenz[a]anthracene plus near-u.v. light initiates DNA damage and repair in Chinese hamster cells.

Earlier results on the photodynamic action of several carcinogenic polycyclic aromatic hydrocarbons, and particularly 7,12-dimethylbenz[a]anthracene (DMBA), have been extended to determine if DMBA + near-u.v. light produces damage to DNA. DMBA by itself (30 min, approximately 24 degrees C) introduces relatively few breaks into genomic DNA. The addition of near-u.v. light, however, inserts large numbers of single-strand breaks in a dose-dependent way. Incubation following exposure initially results in the rapid repair of these breaks. A primary role for DNA damage in photodynamic cell killing is not supported by other observations, however. First, caffeine, an inhibitor of radiation-associated DNA repair processes, has only a minor effect on the oxygen-dependent killing of cells exposed to DMBA + u.v. light. Second, along with the repair of DNA breaks, the insertion of additional breaks becomes evident leading to a massive breakdown of genomic DNA due to an endonucleolytic-like attack. And third, lethally affected cells rapidly lose their surface attachment. Because light induces damage in DNA when DMBA is present, it is likely that DMBA becomes closely associated with DNA. Thus, a starting point for mutagenic and carcinogenic action in the absence of light is suggested although activation of DMBA by a P-450 system does not appear to be a prerequisite of DMBA-DNA association. Still, DNA as such may not be the initial or the primary target for light-induced cell killing. In addition to interacting with DNA, DMBA is sequestered by membranes, suggesting that killing results from an oxygen-dependent release of catabolic enzymes. These enzymes, which may come from lysosomes, degrade DNA but concomitantly release surface-attached cells into the medium.

Determination of Relative Biological Effectiveness (RBE) of Soft X Rays

Differences in clinical results reported with the use of iodine-125 encapsulated implants compared to those with gold-198, radon-222, and iridium-192 prompted a study of the RBE, relative to60 Co γ radiation, of X rays simulating the spectral distribution of radiation from encapsulated125 I. The parameter used for the RBE determination was the killing of surface-attached log-phase HeLa-S3 cells. The RBE was determined at dose rates varying from 15 to 130 rad/min which are about 90 to 500 times greater than those employed in therapy with interstitial ${}^{125}{\rm I} \text{implants}$ . It was found to be approximately 1.4 when measured as a ratio of $D_{0}\text{'}{\rm s}$ and 1.3 as determined on the basis of the ratio of doses for 10% survival (dose rates of 15 and 30 rad/min). The RBE values were found to be dose-rate dependent, increasing linearly from 130 to 30 rad/min and possibly leveling off below this point.

The response of cultured human normal glial cells to growth factors.

This chapter discusses the response of cultured human normal glial cells to growth factors. Normal surface-attached animal cells in culture divide at the maximal rate only as long as free space is available. When the whole vessel surface is covered by cells, proliferation rate decreases, and a stationary phase appears at the terminal cell density. In this study, an assay system for growth factors was developed. Putative growth factors are added to serum-free human glial cell cultures, and the incorporation of tritiated thymidine is determined. The finding of a glial cell-stimulating polypeptide somatomedin B is reported. Fibroblast growth factor (FGF) was found to be a potent glial cell growth promoter. Human osteosarcoma cells in culture, release a growth factor active in the glial cell assay. The factor is nondialyzable, trypsin sensitive, and fairly heat stable. The chapter discusses its possible role in the deficient growth control of the osteosarcoma cells. The major part of the growth-promoting activity of human serum was found to be platelet dependent. Serum prepared from platelet-poor plasma contained less activity than serum from platelet-rich plasma.

Daughter cell repair by mammalian cells in culture after potentially lethal radiation damage.

COLONY formation by irradiated surface-attached mammalian cells in culture requires that at least one of the progeny produced at the first post-irradiation division retains the capacity for unlimited division. Abortive colonies result when the irradiated parent cell has experienced sufficient damage to suppress eventual colony formation, but not to prevent the production of a small number of progeny. We now present data which indicate that, if given a suitable environment, progeny from cells “lethally” damaged by X-radiation (not capable of producing a macroscopic colony) can repair damage transferred (sectored) from the parent cell.