Spreading of mouse fibroblasts on the substrate with multiple spikes

Mouse embryo fibroblasts were cultivated on special substrates with discontinuous surfaces. The substrates were silicon plates with multiple vertical (65-90 microns height) spike-like silicon microcrystals evenly distributed on the plate surfaces. It was shown that the cells were successfully spread and flattened on these substrates. The spread cells formed several discrete attachment zones at the tops and side surfaces of the spikes; these zones were separated from one another by distances considerably greater than the diameter of the unspread cell. At early stages of spreading the unspread cells attached to the tops of single spikes and extended long filopodia attached to the distant spikes. At later stages the lamellae were formed between the filopodia: probably these filopodia served as guidelines for extension of lamellae and progressive cell spreading. These experiments demonstrated that continuity of substrate surface is not a necessary condition for advanced cell spreading.     

Substrate rigidity regulates the formation and maintenance of tissues

The ability of cells to form tissues represents one of the most fundamental issues in biology. However, it is unclear what triggers cells to adhere to one another in tissues and to migrate once a piece of tissue is planted on culture surfaces. Using substrates of identical chemical composition but different flexibility, we show that this process is controlled by substrate rigidity: on stiff substrates, cells migrate away from one another and spread on surfaces, whereas on soft substrates they merge to form tissue-like structures. Similar behavior was observed not only with fibroblastic and epithelial cell lines but also explants from neonatal rat hearts. Cell compaction on soft substrates involves a combination of weakened adhesions to the substrate and myosin II-dependent contractile forces that drive cells toward one another. Our results suggest that tissue formation and maintenance is regulated by differential mechanical signals between cell-cell and cell-substrate interactions, which in turn elicit differential contractile forces and adhesions to determine the preferred direction of cell migration and association.     

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.     

Modeling of shear stress experienced by endothelial cells cultured on microstructured polymer substrates in a parallel plate flow chamber

The application of physical stimuli to cell populations in tissue engineering and regenerative medicine may facilitate significant scientific and clinical advances. However, for the most part, these stimuli are evaluated in isolation, rather than in combination. This study was designed to combine two physical stimuli. The first being a microstructured tissue culture polystyrene substrate, known to produce changes in cell shape and orientation, and the second being laminar shear stress in a parallel plate flow chamber. The combined effects of these stimuli on endothelial cell monolayers cells were evaluated in a parallel plate flow chamber and using a computational fluid dynamics (CFD) model. The topography of the cell monolayers cultured on different microstructured surfaces was determined using confocal laser scanning microscopy (CLSM), and this topographic information was used to construct the CFD model. This research found that while the specific underlying structures were effectively planarized by the cell monolayer, significant differences in cell shape and orientation were observed on the different microstructured surfaces. Cells cultured on grooved substrates aligned in the direction of the grooves and showed higher retention after 1-h LSS conditioning than those cultured on pillars. The modeled shear stress distributions also showed differences. While minor differences in the magnitude of shear stress were noted, aligned cell monolayers experienced significantly lower spatial gradients of shear stress when compared with cells that were not pre-aligned by surface features. The results presented here provide an analysis of how one form of physical stimulus can be moderated by another and also provide a methodology by which the understanding of cell responses to topographic and mechanical stimuli can be further advanced.     

Giardia lamblia attachment force is insensitive to surface treatments

Giardia lamblia cell populations show 90% detachment from glass under normal forces of 2.43+/-0.33 nN applied by centrifugation. Detachment forces were not significantly different for cells attached to positively charged, hydrophobic, or inert surfaces than for cells attached to plain glass. The insensitivity of attachment force to surface treatment is consistent with a suction-based mechanism of attachment.     

Flow around cells adhered to a microvessel wall. I. Fluid stresses and forces acting on the cells

To evaluate the fluid forces acting on cells adhered to a microvessel wall, we numerically studied the flow field around adherent cells and the distribution of the stresses on their surfaces. For simplicity, the cells were modeled as rigid particles attached to a wall of a circular cylindrical tube regularly in the flow direction, in a row or two rows. It was found that not the detailed shape of the model cells but their height from the vessel wall is a key determinant of the fluid forces and torque acting on them. In both arrangements of one row and two rows, the axial spacing between neighboring adherent cells significantly affects the distributions of the stresses on them, which results in drastic variations of the fluid forces with the axial spacing and the relative positions with respect to their neighboring cells. The drag force acting on an adherent cell in the vessel was evaluated to be larger than the value in the 2D chamber flow at the same wall shear stress, mainly due to much larger variations of the pressure distribution on the cell surface in the vessel flow.     

Removal of ligand-bound liposomes from cell surfaces by microbubbles exposed to ultrasound

Gas-filled microbubbles attached to cell surfaces can interact with focused ultrasound to create microstreaming of nearby fluid. We directly observed the ultrasound/microbubble interaction and documented that under certain conditions fluorescent particles that were attached to the surface of live cells could be removed. Fluorescently labeled liposomes that were larger than 500 nm in diameter were attached to the surface of endothelial cells using cRGD targeting to αvβ3 integrin. Microbubbles were attached to the surface of the cells through electrostatic interactions. Images taken before and after the ultrasound exposure were compared to document the effects on the liposomes. When exposed to ultrasound with peak negative pressure of 0.8 MPa, single microbubbles and groups of isolated microbubbles were observed to remove targeted liposomes from the cell surface. Liposomes were removed from a region on the cell surface that averaged 33.1 μm in diameter. The maximum distance between a single microbubble and a detached liposome was 34.5 μm. Single microbubbles were shown to be able to remove liposomes from over half the surface of a cell. The distance over which liposomes were removed was significantly dependent on the resting diameter of the microbubble. Clusters of adjoining microbubbles were not seen to remove liposomes. These observations demonstrate that the fluid shear forces generated by the ultrasound/microbubble interaction can remove liposomes from the surfaces of cells over distances that are greater than the diameter of the microbubble.     

Atomic force microscopy investigation of fibroblasts infected with wild-type and mutant murine leukemia virus (MuLV)

NIH 3T3 cells were infected in culture with the oncogenic retrovirus, mouse leukemia virus (MuLV), and studied using atomic force microscopy (AFM). Cells fixed with glutaraldehyde alone, and those postfixed with osmium tetroxide, were imaged under ethanol according to procedures that largely preserved their structures. With glutaraldehyde fixation alone, the lipid bilayer was removed and maturing virions were seen emerging from the cytoskeletal matrix. With osmium tetroxide postfixation, the lipid bilayer was maintained and virions were observable still attached to the cell surfaces. The virions on the cell surfaces were imaged at high resolution and considerable detail of the arrangement of protein assemblies on their surfaces was evident. Infected cells were also labeled with primary antibodies against the virus env surface protein, followed by secondary antibodies conjugated with colloidal gold particles. Other 3T3 cells in culture were infected with MuLV containing a mutation in the gPr80(gag) gene. Those cells were observed by AFM not to produce normal MuLV on their surfaces, or at best, only at very low levels. The cell surfaces, however, became covered with tubelike structures that appear to result from a failure of the virions to properly undergo morphogenesis, and to fail in budding completely from the cell's surfaces.     

On the mechanism of internalization of opsonized particles by rat Kupffer cells in vitro

The attachment and internalization of opsonized sheep red blood cells by cultured rat Kupffer cells were studied with phase-contrast and scanning electron microscopy (SEM) as well as timelapse microcinematography. We observed that sheep red cells coated with IgG attached over the entire Kupffer cell surface at random, whereas those coated with IgM and complement attached all over the cell with the exception of the extreme periphery. When the Fc and C₃ receptors were given appropriate stimuli to internalize the attached red cells, they functioned very differently. In Fc internalization, the Kupffer cell membrane rose above the main cell body and wrapped tightly around the attached red cell, eventually surrounding it entirely. In the C₃ internalization, triggered by new-born calf serum, the membrane activity was less spectacular; the folds that did sometimes rise up were coarser and did not fit tightly around the red cell, which was eventually interiorized by a sinking, deep into the cytoplasm of the Kupffer cell. These two mechanisms of internalization also showed different sensitivities to cytochalasin B (CB); the Fc internalization being far more vulnerable to this inhibitor of microfilament activities. Studies with colchicine, however, did not show any clear-cut difference in sensitivity between the two cases.     

Microjet impingement followed by scanning electron microscopy as a qualitative technique to compare cellular adhesion to various biomaterials

Adhesion of cells to biomaterial surfaces is one of the major factors which mediates their biocompatibility. Quantitative or qualitative cell adhesion measurements would be useful for screening new implant materials. Microjet impingement has been evaluated by scanning electron microscopy, to determine to what extent it measures cell adhesion. The shear forces of the impingement, on the materials tested here, are seen to be greater than the cohesive strength of the cells in the impinged area, causing their rupture. The cell bodies are removed during impingement, leaving the sites of adhesion and other cellular material behind. Thus the method is shown not to provide quantification of cell adhesion forces for the metals and culture plastic tested. It is suggested that with highly adherent biomaterials, the distribution and patterns of these adhesion sites could be used for qualitative comparisons for screening of implant surfaces.     

The hemodynamic forces acting on thrombi, from incipient attachment of single cells to maturity and embolization

We consider the steady fluid forces acting on a thrombus from the time of first contact of a single cell with a natural or artificial surface, through the attachment process and growth to embolization. For a hemi-spherical or cylindrical attached cell of height less than 1/100-1/20th of the channel width, shear and tensile stresses are solely dependent on viscosity and on the ratio of average fluid velocity to channel width vt/Dt (shear rate). Large values of this ratio reduce adhesion and increase embolization. The average shear stress on such cells is approximately 1-10 Pa (10-100 dyn cm2), the average tensile stress about three times higher. For other shapes and larger protrusions, stress varies with protrusion height as well. Maturing thrombi composed of cell aggregates embedded in a fibrin mesh do not appear to allow significant fluid flow through their porous structure. The interior forces are then due solely to hydrostatic pressure and initially vary directly with vt/Dt and inversely with thrombus height Hp, thus favouring embolization at an early stage and in arterial systems. Rough surfaces are identified as causing an increase in dwell-time and possibly immobilizing an unattached cell due to 'negative lift'.     

Development of surface adhesion in Caulobacter crescentus

Caulobacter crescentus has a dimorphic life cycle composed of a motile stage and a sessile stage. In the sessile stage, C. crescentus is often found tightly attached to a surface through its adhesive holdfast. In this study, we examined the contribution of growth and external structures to the attachment of C. crescentus to abiotic surfaces. We show that the holdfast is essential but not sufficient for optimal attachment. Rather, adhesion in C. crescentus is a complex developmental process. We found that the attachment of C. crescentus to surfaces is cell cycle regulated and that growth or energy or both are essential for this process. The initial stage of attachment occurs in swarmer cells and is facilitated by flagellar motility and pili. Our results suggest that strong attachment is mediated by the synthesis of a holdfast as the swarmer cell differentiates into a stalked cell.     

Effect of Ionic Strength on Initial Interactions of Escherichia coli with Surfaces, Studied On-Line by a Novel Quartz Crystal Microbalance Technique

A novel quartz crystal microbalance (QCM) technique was used to study the adhesion of nonfimbriated and fimbriated Escherichia coli mutant strains to hydrophilic and hydrophobic surfaces at different ionic strengths. This technique enabled us to measure both frequency shifts (Deltaf), i.e., the increase in mass on the surface, and dissipation shifts (DeltaD), i.e., the viscoelastic energy losses on the surface. Changes in the parameters measured by the extended QCM technique reflect the dynamic character of the adhesion process. We were able to show clear differences in the viscoelastic behavior of fimbriated and nonfimbriated cells attached to surfaces. The interactions between bacterial cells and quartz crystal surfaces at various ionic strengths followed different trends, depending on the cell surface structures in direct contact with the surface. While Deltaf and DeltaD per attached cell increased for nonfimbriated cells with increasing ionic strengths (particularly on hydrophobic surfaces), the adhesion of the fimbriated strain caused only low-level frequency and dissipation shifts on both kinds of surfaces at all ionic strengths tested. We propose that nonfimbriated cells may get better contact with increasing ionic strengths due to an increased area of contact between the cell and the surface, whereas fimbriated cells seem to have a flexible contact with the surface at all ionic strengths tested. The area of contact between fimbriated cells and the surface does not increase with increasing ionic strengths, but on hydrophobic surfaces each contact point seems to contribute relatively more to the total energy loss. Independent of ionic strength, attached cells undergo time-dependent interactions with the surface leading to increased contact area and viscoelastic losses per cell, which may be due to the establishment of a more intimate contact between the cell and the surface. Hence, the extended QCM technique provides new qualitative information about the direct contact of bacterial cells to surfaces and the adhesion mechanisms involved.     

Assessment of cell-substrate adhesion by a centrifugal method

Summary A centrifugal method has been evaluated for measuring the strength of Vero Green Monkey kidney cell adhesion to growth surfaces. The centrifugal force necessary to remove cells gave a quantitative measure of cell adhesion and hence the quality of the growth surface. After being subjected to high gravity forces, both the remaining attached cells and the detached cells were viable, indicating the detachment process did not simply rupture the cell. Electron microscope examination of growth surfaces after cell detachment suggested that remnants related to filopodia remained.     

Contact behaviour of setal tips in the hairy attachment system of the fly Calliphora vicina (Diptera, Calliphoridae): a cryo-SEM approach

The fly Calliphora vicina (Diptera, Calliphoridae) bears attachment pads (pulvilli) covered with setae on their ventral sides. These structures enable attachment to smooth vertical surfaces and ceilings. The contact between the terminal setal tips (spatulae) and various substrates was visualised using various experimental techniques combined with conventional scanning electron microscopy (SEM) and cryo-SEM. The results show that the setal endplates are highly flexible structures that form contact with the surface by bending their tips in the distal direction. With conventional SEM, a comparison of partly attached endplates with unattached endplates demonstrated the presence of a distinct marginal bulge. As observed with cryo-SEM, the bulge continuously disappeared as a larger area of the endplate came into contact. Two explanations of this result are suggested. First, the volume between the bulge, the mid-part of the endplate and the substrate may be filled with a fluid secretion that is released into the contact area in the endplate region. Second, the flexible central part of the endplate may jump into contact with the substrate during contact formation.     

The influence of cell shape on the induction of functional differentiation in mouse mammary cells in vitro

Summary To define more clearly the in vitro conditions permissive for hormonal induction of functional differentiation, we cultured dissociated normal mammary cells from prelactating mice in or on a variety of substrates. Cultivation of an enriched epithelial cell population in association with living adult mammary stroma in the presence of lactogenic hormones resulted in both morphological and biochemical differentiation. This differentiation, however, was not enhanced over that seen when the cells were associated with killed stroma, provided that the killed stroma had a flexibility similar to that of the living stroma. Cells cultured in inflexible killed stroma usually did not differentiate. Cells cultured within the flexible environment of a collagen gel, but removed from the gas-medium interface, differentiated in a manner similar to those cultured in flexible stroma. Cells cultured on the surface of an attached collagen gel were squamous, and their basolateral surfaces were sequestered from the medium; they did not differentiate. Cells cultured on floating collagen gels were cuboidal-columnar, with basolateral surfaces exposed to the medium, and showed good functional differentiation. Cells cultured on inflexible floating collagen gels were extremely flattened and had exposed basolateral surfaces, and showed no evidence of functional differentiation. We infer that assumption of cuboidal to columnar shapes similar to those of mammary cells in vivo may be important to the induction of functional differentiation in vitro. The additional requirement of basolateral cell surface exposure also is important. This work was supported by U.S. Public Health Service Grants CA-05045 and CA-09041 from the National Cancer Institute, Bethesda, MD.     

Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy

Cells of many kinds adhere firmly to glass or plastic surfaces which have been pretreated with polylysine. The attachment takes place as soon as the cells make contact with the surfaces, and the flattening of the cells against the surfaces is quite rapid. Cells which do not normally adhere to solid surfaces, such as sea urchin eggs, attach as well as cells which normally do so, such as amebas or mammalian cells in culture. The adhesion is interpreted simply as the interaction between the polyanionic cell surfaces and the polycationic layer of adsorbed polylysine. The attachment of cells to the polylysine-treated surfaces can be exploited for a variety of experimental manipulations. In the preparation of samples for scanning or transmission electron microscopy, the living material may first be attached to a polylysine- coated plate or grid, subjected to some experimental treatment (fertilization of an egg, for example), then transferred rapidly to fixative and further passed through processing for observation; each step involves only the transfer of the plate or grid from one container to the next. The cells are not detached. The adhesion of the cell may be so firm that the body of the cell may be sheared away, leaving attached a patch of cell surface, face up, for observation of its inner aspect. For example, one may observe secretory vesicles on the inner face of the surface (3) or may study the association of filaments with the inner surface (Fig. 1). Subcellular structures may attach to the polylysine-coated surfaces. So far, we have found this to be the case for nuclei isolated from sea urchin embryos and for the microtubules of flagella, which are well displayed after the membrane has been disrupted by Triton X-100 (Fig. 2).     

The unfolding of the P pili quaternary structure by stretching is reversible, not plastic

P pili are protein filaments expressed by uropathogenic Escherichia coli that mediate binding to glycolipids on epithelial cell surfaces, which is a prerequisite for bacterial infection. When a bacterium, attached to a cell surface, is exposed to external forces, the pili, which are composed of approximately 10(3) PapA protein subunits arranged in a helical conformation, can elongate by unfolding to a linear conformation. This property is considered important for the ability of a bacterium to withstand shear forces caused by urine flow. It has hitherto been assumed that this elongation is plastic, thus constituting a permanent conformational deformation. We demonstrate, using optical tweezers, that this is not the case; the unfolding of the helical structure to a linear conformation is fully reversible. It is surmised that this reversibility helps the bacteria regain close contact to the host cells after exposure to significant shear forces, which is believed to facilitate their colonization.     

The invasion of HeLa cells by Salmonella typhimurium: reversible and irreversible bacterial attachment and the role of bacterial motility

The interactions which brought about the invasion of HeLa cells by Salmonella typhimurium consisted of a sequence of three phases. Initially, the motility of the bacteria facilitated their contact with the HeLa cells whereupon the bacteria became attached in a reversible manner (i.e. the bacteria could be removed readily by washing the HeLa cell monolayers with Hanks' Balanced Salt solution). The binding forces responsible for reversible attachment were probably the weak long-range forces of the secondary minimum level of attractive interactions between the bacterium and the HeLa cell. Reversible attachment was a necessary interlude before the bacteria became irreversibly attached to the surfaces of the HeLa cells (i.e. the bacteria were no longer removed by the washing procedure that removed the reversibly attached salmonellae). Irreversible attachment was prevented in solutions of low ionic strength; the forces responsible were probably those of the primary minimum generated between the HeLa cell and a bacterial adhesion which was capable of acting over only short distances between the reversibly attached bacterium and the HeLa cell (i.e. probably less than 15 nm). Only irreversibly attached bacteria proceeded to the third phase and were internalized by the HeLa cells.     

Fluid mechanics of the zebrafish embryonic heart trabeculation

Embryonic heart development is a mechanosensitive process, where specific fluid forces are needed for the correct development, and abnormal mechanical stimuli can lead to malformations. It is thus important to understand the nature of embryonic heart fluid forces. However, the fluid dynamical behaviour close to the embryonic endocardial surface is very sensitive to the geometry and motion dynamics of fine-scale cardiac trabecular surface structures. Here, we conducted image-based computational fluid dynamics (CFD) simulations to quantify the fluid mechanics associated with the zebrafish embryonic heart trabeculae. To capture trabecular geometric and motion details, we used a fish line that expresses fluorescence at the endocardial cell membrane, and high resolution 3D confocal microscopy. Our endocardial wall shear stress (WSS) results were found to exceed those reported in existing literature, which were estimated using myocardial rather than endocardial boundaries. By conducting simulations of single intra-trabecular spaces under varied scenarios, where the translational or deformational motions (caused by contraction) were removed, we found that a squeeze flow effect was responsible for most of the WSS magnitude in the intra-trabecular spaces, rather than the shear interaction with the flow in the main ventricular chamber. We found that trabecular structures were responsible for the high spatial variability of the magnitude and oscillatory nature of WSS, and for reducing the endocardial deformational burden. We further found cells attached to the endocardium within the intra-trabecular spaces, which were likely embryonic hemogenic cells, whose presence increased endocardial WSS. Overall, our results suggested that a complex multi-component consideration of both anatomic features and motion dynamics were needed to quantify the trabeculated embryonic heart fluid mechanics.