Method of preparing suspended cells for scanning electron microscopy]

To preserve the lifetime morphology of the surface of suspended cells, these must be fixed in suspensions. The subsequent stages of cell preparation for scanning electron microscopy (dehydratation, critical point drying, coating) are considerably facilitated if fixed cells are preliminary attached to some substrate surface. An effective substrate should provide a firm rather than selective attachment of the overwhelming majority of fixed cells; the substrate should be also available for a wide application. The trial of different types of substrates showed a sufficient effectivity of plates made of commercial aluminium foil. In tests with murine embryonal and transformed fibroblasts as well as with human blood leukocytes, in average 90 per cent of cells fixed with glutaraldehyde in suspensions got attached to foil substrate surfaces; the fixed cells both settled from suspension and attached were seen distributed evenly on the substrate surface. The use of aluminium foil substrates made it possible to study the surface topography of some types of suspended cells.     

Early adhesion of human mesenchymal stem cells on TiO(2) surfaces studied by single-cell force spectroscopy measurements

Understanding the interactions involved in the adhesion of living cells on surfaces is essential in the field of tissue engineering and biomaterials. In this study, we investigate the early adhesion of living human mesenchymal stem cells (hMSCs) on flat titanium dioxide (TiO(2) ) and on nanoporous crystallized TiO(2) surfaces with the use of atomic force microscopy-based single-cell force spectroscopy measurements. The choice of the substrate surfaces was motivated by the fact that implants widely used in orthopaedic and dental surgery are made in Ti and its alloys. Nanoporous TiO(2) surfaces were produced by anodization of Ti surfaces. In a typical force spectroscopy experiment, one living hMSC, immobilized onto a fibronectine-functionalized tipless lever is brought in contact with the surface of interest for 30 s before being detached while recording force-distance curves. Adhesion of hMSCs on nanoporous TiO(2) substrates having inner pore diameter of 45 nm was lower by approximately 25% than on TiO(2) flat surfaces. Force-distance curves exhibited also force steps that can be related to the pulling of membrane tethers from the cell membrane. The mean force step was equal to 35 pN for a given speed independently of the substrate surface probed. The number of tethers observed was substrate dependent. Our results suggest that the strength of the initial adhesion between hMSCs and flat or nanoporous TiO(2) surfaces is driven by the adsorption of proteins deposited from serum in the culture media.     

Generation of easily accessible human kidney tubules on two‐dimensional surfaces in vitro

The generation of tissue‐like structures in vitro is of major interest for various fields of research including in vitro toxicology, regenerative therapies and tissue engineering. Usually 3D matrices are used to engineer tissue‐like structures in vitro, and for the generation of kidney tubules, 3D gels are employed. Kidney tubules embedded within 3D gels are difficult to access for manipulations and imaging. Here we show how large and functional human kidney tubules can be generated in vitro on 2D surfaces, without the use of 3D matrices. The mechanism used by human primary renal proximal tubule cells for tubulogenesis on 2D surfaces appears to be distinct from the mechanism employed in 3D gels, and tubulogenesis on 2D surfaces involves interactions between epithelial and mesenchymal cells. The process is induced by transforming growth factor‐β₁, and enhanced by a 3D substrate architecture. However, after triggering the process, the formation of renal tubules occurs with remarkable independence from the substrate architecture. Human proximal tubules generated on 2D surfaces typically have a length of several millimetres, and are easily accessible for manipulations and imaging, which makes them attractive for basic research and in vitro nephrotoxicology. The experimental system described also allows for in vitro studies on how primary human kidney cells regenerate renal structures after organ disruption. The finding that human kidney cells organize tissue‐like structures independently from the substrate architecture has important consequences for kidney tissue engineering, and it will be important, for instance, to inhibit the process of tubulogenesis on 2D surfaces in bioartificial kidneys.     

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.     

Macrophages form circular zones of very close apposition to IgG-coated surfaces

When phagocytes spread on surfaces coated with ligands such as IgG, they form a tight seal with the substrate. This seal excludes soluble macromolecules in the medium from the interface between the cell and substrate. In contrast, when cells spread on control surfaces that are not coated with ligands, the underside of the cell remains freely accessible to soluble proteins (Wright and Silverstein: Nature 309:359, 1984). We employed reflection-interference microscopy (RIM) to determine where the seal forms during interaction with ligand (IgG)-coated surfaces. Human monocyte-derived macrophages (MO) were plated at 37 degrees C on dinitrophenylated (DNP)-glass coverslips (control substrate), IgM anti-DNP-DNP-coated glass (control substrate), or on IgG anti-DNP-DNP-coated glass (phagocytosis-promoting substrate). Live or fixed cells were examined by RIM. Spreading on control surfaces at 37 degrees C was complete in 25 minutes, whereas spreading on IgG-coated surfaces was maximal within 15 minutes and resulted in cell-substrate contact area 1.6 X that of control cells. Within 1 h at 37 degrees C, 90% of MO that spread on IgG-coated substrates, but not on control substrates, excluded macromolecules from their underside. A minor population of cells (19%) exhibited a uniform iron gray RIM appearance indicating an even, close approach to the substrate. These cells may represent early stages of frustrated phagocytosis. In contrast to cells on control substrates, 70% of cells on IgG-coated substrates developed continuous peripheral dark rings in RIM indicative of close association with the substrate. Essentially all cells with peripheral dark rings in RIM excluded macromolecules from their underside. Enclosed within this ring was an area of greater separation between the cell membrane and the substrate, as indicated by the lighter grey of this region in RIM and by the accessibility of substrate to anti-substrate antibody when breaks in the dark ring occur. Thus, MO can create a closed compartment between plasma membrane and substrate that excludes proteins in the surrounding medium, thereby protecting substances secreted into this space from potentially inhibitory substances in the medium.     

Antimicrobial testing for surface‐immobilized agents with a surface‐separated live–dead staining method

Modification of a traditional live–dead staining technique based on fluorescence microscopy has yielded an improved method capable of differentiating surface‐immobilized antimicrobial agents from those agents acting via solution diffusion processes. By utilizing an inoculation chamber comprised of 50 µm polystyrene spheres as spacers between test substrate and coverslip control surfaces, three distinct bacterial cell populations can be probed by fluorescence microscopy for antimicrobial activity: (1) cells adhered to the coverslip, (2) cells adhered to the substrate, and (3) mobile cells in solution. Truly immobilized antimicrobial agents were found efficacious only at the substrate surface, while elutable agents were effective against all three populations. Glass surfaces derivatized with either quaternized poly dimethylaminoethylmethacrylate (pDMAEMA) or 3‐(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (Si‐QAC) were compared with bare glass control surfaces after contact and 4 h incubation with Staphylococcus aureus. pDMAEMA surfaces were both antimicrobial and immobilized, whereas the Si‐QAC surfaces were only observed to be antimicrobial via active diffusion. In contrast to conventional thinking, Si‐QAC surfaces showed no kill after removing all Si‐QAC elutables via rinsing procedures. The semi‐quantitative surface‐separated live–dead staining (SSLDS) technique provides mechanistic insight and represents a significant improvement relative to current microbiological test methods for evaluating immobilized, antimicrobial agents. Biotechnol. Bioeng. 2011; 108:231–236. © 2010 Wiley Periodicals, Inc.     

Electron microscopic observations of the surface of L-cells in culture

Summary Techniques are described for the preparation of preshadowed replicas of both the upper and lower surfaces of L-cells in culture, and of cross sections of L-cells growing on a cellophane substrate. These revealed long slender microvilli, 800 to 1,100 A in diameter, projecting from both upper and lower surfaces of the cells. These microvilli were frequently observed to contact other cells and substrate, and to leave material behind on the substrate. The plasma membrane of the lower surface was separated from the substrate by an electron-lucent gap 200 to 300 A wide. The surface coat of the L-cell was visualized by staining with colloidal iron and ruthenium. Staining with colloidal iron was most intense on the surface of the microvilli. The gap between cell and substrate was intensely stained with ruthenium red. Enzymatic digestion of living cells revealed that both trypsin and neuraminidase reduced the staining of the cell coat by colloidal iron, whereas only trypsin altered its staining with ruthenium red. After trypsin treatment, fragments of an amorphous material with the staining characteristics of the cell coat were observed between the denuded cells. Treatment with ribonuclease, chymotrypsin or hyaluronidase did not affect the staining of the cell coat.     

Cellular responses to substrate topography: role of myosin II and focal adhesion kinase

Although two-dimensional cultures have been used extensively in cell biological research, most cells in vivo exist in a three-dimensional environment with complex topographical features, which may account for at least part of the striking differences between cells grown in vivo and in vitro. To investigate how substrate topography affects cell shape and movement, we plated fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars. Compared to cells on flat surfaces, 3T3 cells on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability. These responses may be attributed to stabilization of cell adhesion on pillars coupled to myosin II-dependent contractions toward pillars. Moreover, using FAK-/- fibroblasts we showed that focal adhesion kinase, or FAK, is essential for the responses to substrate topography. We propose that increased surface contact provided by topographic features guides cell migration by regulating the strength of local adhesions and contractions, through a FAK- and myosin II-dependent mechanism.     

In search of the microbe/mineral interface: quantitative analysis of bacteria on metal surfaces using vertical scanning interferometry

To understand the development of biofilms on metal surfaces, analysis of initial bacterial attachment to surfaces is crucial. Here we present the results of a study, using Shewanella oneidensis MR-1 as a model organism, in which vertical scanning interferometry (VSI) was used to investigate the initial stages of cell attachment to glass, steel and aluminium surfaces. It was found that while VSI gave unambiguous results with opaque surfaces, when reflective surfaces were used, an artifact sometimes appeared, with the bacteria appearing as rod-shaped pits rather than as cells on the surface. When the bacteria were altered to increase opacity, this artifact disappeared, and upon further investigation, it was found that the observational artifact was the result of a conflict between light reflected from the bacteria and the light reflected from the bacteria–metal interface. These results suggest that not only can bacteria be measured on surfaces using VSI, but with some modifications to the analytical software, there may be a unique window for studying the bacterial/substrate interface that can be used for quantitative observations. Imaging and characterization of the bacteria–substrate interface in vivo (previously invisible) will provide new insights into the interactions that occur at this important juncture.     

Surface modification of silicon and gold-patterned silicon surfaces for improved biocompatibility and cell patterning selectivity

Clean silicon and gold-patterned silicon platforms were modified with methoxy-polyethylene glycol (M-PEG silane) via a self-assembly technique, which significantly improved their plasma protein resistance capability and cell patterning selectivity. Fibrinogen and IgG were used as model plasma proteins to study the efficacy of PEG layers in resisting protein adsorption. Selective cell patterning on the gold regions of a gold-patterned silicon substrate and tissue compatibility were studied with macrophage and fibroblast cells. The research also revealed how the presence of gold electrodes on a silicon substrate would influence the cell patterning selectivity. Our experimental results showed that the PEG-modified silicon surfaces had a high resistivity to protein and cell attachment and that the PEG-modified gold-patterned silicon surfaces nearly completely eliminated the protein adsorption and cell attachment on silicon. This study provides a new approach to developing biocompatible surfaces for silicon-based BioMEMS devices, particularly for biosensors where a metal-insulator format must be enforced.     

The effect of substrate and adsorbed proteins on adhesion, growth and shape of CaCo-2 cells

Extracellular matrix (ECM) proteins play a critical role in many cellular functions, from spreading, migration and proliferation to apoptosis. This role can be altered when proteins of the native ECM are adsorbed to different substrates which cause structural modifications that can influence their biological function. The effects on CaCo-2 cells of laminin-1, fibronectin, collagen-1 and ECM gel adsorbed to glass and to tissue culture polystyrene (PS) were compared in terms of adhesion, proliferation, shapes and spreading of cells in culture. Significant differences between glass and PS surfaces were observed for proliferation and cell shape. Protein surfaces prepared on PS substrates had, in most cases, more pronounced effects on cells than uncoated PS, especially if coated by collagen-1. Adsorbed ECM gel was the most adhesive for cells, but its effect on cell proliferation was not notably different from the controls (glass or PS). These findings indicate that the choice of the substrate can have a significant effect on experimental results and should be taken into consideration when comparing results obtained on different surfaces.     

Ammonia plasma treatment of polystyrene surfaces enhances proliferation of primary human mesenchymal stem cells and human endothelial cells

The control of surface properties is a substantial step in the development and improvement of biomaterials for clinical applications as well as for their use in tissue engineering. Interaction of the substrate surface with the biochemical or biological environment is crucial for the outcome of the applied biomaterial and therefore should meet specific requirements regarding the chemical composition, wettability, elasticity, and charge. In this study, we examined the effect of chemical groups introduced by low pressure plasma treatments of polystyrene surfaces on the cell behavior of primary human mesenchymal stem cells (hMSCs) and dermal microvascular endothelial cells (hDMECs). X-ray photoelectron spectroscopy analysis and contact angle measurements were employed to evaluate ammonia-, carbon dioxide-, and acrylic acid-plasma modifications to substrate surfaces. HMSCs and hDMECs were analyzed simultaneously to identify the most suitable surface functionalization for each cell type. Significantly higher cell proliferation was detected on ammonia plasma-treated surfaces. Cell-material interaction could be shown on all created interfaces as well as the expression of typical cell markers. Hence, the applied plasma treatment presents a suitable tool to improve culture condition on polystyrene for two important cell types (hMSCs and hDMECs) in the field of tissue engineering.     

Guidance of Cell Migration by Substrate Dimension

There is increasing evidence to suggest that physical parameters, including substrate rigidity, topography, and cell geometry, play an important role in cell migration. As there are significant differences in cell behavior when cultured in 1D, 2D, or 3D environments, we hypothesize that migrating cells are also able to sense the dimension of the environment as a guidance cue. NIH 3T3 fibroblasts were cultured on micropatterned substrates where the path of migration alternates between 1D lines and 2D rectangles. We found that 3T3 cells had a clear preference to stay on 2D rather than 1D substrates. Cells on 2D surfaces generated stronger traction stress than did those on 1D surfaces, but inhibition of myosin II caused cells to lose their sensitivity to substrate dimension, suggesting that myosin-II-dependent traction forces are the determining factor for dimension sensing. Furthermore, oncogene-transformed fibroblasts are defective in mechanosensing while generating similar traction forces on 1D and 2D surfaces. Dimension sensing may be involved in guiding cell migration for both physiological functions and tissue engineering, and for maintaining normal cells in their home tissue.     

Changes in micronucleus frequency resulting from preirradiation of cell culture surfaces

We have initiated a series of experiments to quantify the impact of environmental variables on the observed frequency of micronuclei in monolayer cultures. In this paper the influence of preirradiation of cell culture vessels on micronucleus formation in Chinese hamster ovary cells was examined. Dry cell culture vessels were preirradiated with 2 Gy of either alpha particles or X rays and immediately plated with nonirradiated cells. About 48 h later a group of randomly chosen containers was set aside, and the rest of the containers were exposed to a range of doses of X rays or alpha-particle radiation. Nonirradiated cells plated on previously irradiated cell culture surfaces manifested nearly as many micronuclei as the irradiated cells. In all experiments, preirradiation of the cell substrate (the culture dish) led to a significantly increased micronucleus frequency relative to unirradiated substrate. These results suggest that methods of cell culture vessel sterilization and the composition of cell attachment surfaces could be a confounding factor, particularly in low-dose experiments.     

Ecological implications of glucosyltransferase phase variation in Streptococcus gordonii.

When sucrose is provided as a substrate for glucosyltransferase (GTF), Spp+ cells of the oral bacteria Streptococcus gordonii grow embedded in an insoluble glucan mass associated with surfaces. Spp- phase variants with lower GTF activity, which either arise from or are grown with Spp+ cells, segregate preferentially as unattached cells in the culture supernatants. Conversely, Spp+ revertants preferentially accumulate on surfaces. GTF phase variation, therefore, may facilitate the dispersion of S. gordonii cells throughout the oral cavity.     

Ecological implications of glucosyltransferase phase variation in Streptococcus gordonii.

When sucrose is provided as a substrate for glucosyltransferase (GTF), Spp+ cells of the oral bacteria Streptococcus gordonii grow embedded in an insoluble glucan mass associated with surfaces. Spp- phase variants with lower GTF activity, which either arise from or are grown with Spp+ cells, segregate preferentially as unattached cells in the culture supernatants. Conversely, Spp+ revertants preferentially accumulate on surfaces. GTF phase variation, therefore, may facilitate the dispersion of S. gordonii cells throughout the oral cavity.     

Interaction of different types of epithelium in a mixed culture

Interaction of several lines of epithelial cells was studied in a mixed culture: FBT (bovine fetal trachea), MDSK (dog kidney), IAR-2 (rat liver), MPTR (SV-40 transformed mouse kidney). During mixed cultivation epithelia cells of different types were capable to force out each other from the substrate to full elimination. This capacity correlated with the pattern of cell contacts with the substrate. It is supposed that epithelial cells can form lamellae at the lower surfaces competing for substrate. Those cells which have lamellae with continuous marginal focal contacts and numerous focal contacts eliminate the cells having lamellae with few focal contacts.     

Photoelectron imaging of viruses and DNA: evaluation of substrates by unidirectional low angle shadowing and photoemission current measurements.

Photoelectron imaging (photoelectron emission microscopy, PEM or PEEM) is a promising high resolution surface-sensitive technique for biophysical studies. At present, image quality is often limited by the underlying substrate. For photoelectron imaging, the substrate must be electrically conductive, low in electron emission, and relatively flat. A number of conductive substrate materials with relatively low electron emission were examined for surface roughness. Low angle, unidirectional shadowing of the specimens followed by photoelectron microscopy was found to be an effective way to test the quality of substrate surfaces. Optimal results were obtained by depositing approximately 0.1 nm of platinum-palladium (80:20) at an angle of 3 degrees. Among potential substrates for photoelectron imaging, silicon and evaporated chromium surfaces were found to be much smoother than evaporated magnesium fluoride, which initially appeared promising because of its very low electron emission. The best images were obtained with a chromium substrate coated with a thin layer of dextran derivatized with spermidine, which facilitated the spreading and adhesion of biomolecules to the surfaces. Making use of this substrate, improved photoelectron images are reported for tobacco mosaic virus particles and DNA-recA complexes.     

Yeast cell adhesion on oligopeptide modified surfaces

Self-assembling oligopeptides are novel materials with potential bioengineering applications; this paper explores the use of one of these oligopeptides, EAK 16 II, for modifying the surface properties of cell-supporting substrates. To characterize the surface properties, thermodynamic measurements of liquid contact angle and surface free energy were correlated to atomic force microscopy (AFM) observations. A critical concentration of 0.1 mg/ml was found necessary to completely modify the surface properties of the substrate with EAK 16 II. Adhesion of a yeast cell, Candida utilis, was modified by the coating of EAK 16 II on both hydrophobic (plastic) and hydrophilic (glass) surfaces: Cell coverage was slightly enhanced on the glass substrate, but decreased significantly on the plastic substrate. This indicates that the yeast cell adhesion was mainly determined via hydrophobic interactions between the substrate and the cell wall. However, on the EAK 16 II modified glass substrate, surface roughness might be a factor in causing a slightly larger cell adhesion than that on bare glass. The morphology of adhered cells was also obtained with AFM imaging, showing a depression at the center of the cell on all substrates. Small depressions on the oligopeptide-coated surfaces and plastic substrate may indicate good water-retaining ability by the cell. There was no apparent difference in cell adhesion and morphology among cells obtained from lag, exponential and stationary growth phases.     

Isolation and characterization of flat cells, a subpopulation of the embryonic chick retina

When the embryonic neutral retina is dissociated into single cells which are maintained in stationary culture, the neuronal cells associate on the surfaces of a second population which we refer to as flat cells. The flat cells appear in the culture in significant numbers after 2 days and are required for neuronal cell attachment. We have been able to isolate pure flat cells from early cultures of mixed retina cells and have identified several antigens which support the concept that these cells are related to the glia. The cells have been tested by immunofluorescence for glial fibrillary acidic protein and have been found positive. Cell surfaces were labeled by transfer of tritiated galactose from UDP-galactose to endogenous acceptors in the presence of exogenous galactosyl transferase. After SDS-PAGE and fluorography, the surface glycoproteins of flat cells were seen to be significantly different from those of the original retina, and from chick fibroblasts. Immunoelectron microscope studies of detergent-extracted flat cells have demonstrated a complex network of intermediate filaments and actin fibers. We conclude that the flat cells are derived from the glia subpopulation of the retina and have adapted to the tissue culture environment by assuming this configuration. The unique surface properties of flat cells may be related to their role as an intermediate substrate between the neuronal cells and the tissue culture dish.