Quantitative Study of the Elastic Modulus of Loosely Attached Cells in AFM Indentation Experiments

When measuring the elastic (Young’s) modulus of cells using AFM, good attachment of cells to a substrate is paramount. However, many cells cannot be firmly attached to many substrates. A loosely attached cell is more compliant under indenting. It may result in artificially low elastic modulus when analyzed with the elasticity models assuming firm attachment. Here we suggest an AFM-based method/model that can be applied to extract the correct Young’s modulus of cells loosely attached to a substrate. The method is verified by using primary breast epithelial cancer cells (MCF-7) at passage 4. At this passage, approximately one-half of cells develop enough adhesion with the substrate to be firmly attached to the substrate. These cells look well spread. The other one-half of cells do not develop sufficient adhesion, and are loosely attached to the substrate. These cells look spherical. When processing the AFM indentation data, a straightforward use of the Hertz model results in a substantial difference of the Young’s modulus between these two types of cells. If we use the model presented here, we see no statistical difference between the values of the Young’s modulus of both poorly attached (round) and firmly attached (close to flat) cells. In addition, the presented model allows obtaining parameters of the brush surrounding the cells. The cellular brush observed is also statistically identical for both types of cells. The method described here can be applied to study mechanics of many other types of cells loosely attached to substrates, e.g., blood cells, some stem cells, cancerous cells, etc.     

Degree of cell spreading on a substrate as the factor determining the nature of cell microrelief in suspension

Quantitative ratio of various types of cell surface microrelief was determined in suspensions prepared from mouse monolayer cultures of embryo fibroblasts grown on different solid substrates: with high (Falcon) or low poly(2-hydroxyethylmethacrylate) adhesiveness; with flat or cylindrical (53-mu curvature radius) surfaces (polyvinylchloride). The electron microscopy revealed that poorly spread cells (on low adhesive or cylindrical substrata) in suspensions had the microvillous surface relief much more often than the cells brought to suspension from highly adhesive or flat substrata. Thus, the lower the degree of cell spreading on the substratum, the higher the probability for the cell to acquire the microvillous relief in suspended state. The microvillous relief of transformed cells in suspensions is, probably, due to their poor spreading on substrata in the monolayer cultures.     

A theoretical derivation of the Contois equation for kinetic modeling of the microbial degradation of insoluble substrates

Although developed as an empirical model to describe microbial growth on soluble substrates, the Contois equation has been widely accepted for kinetic modeling of insoluble substrate degradation. Yet, the mechanistic basis underlining these successful applications remains unanswered. Unlike soluble substrates that mainly cultivate suspended cultures, microbes cultivated on insoluble substrates have the populations that attach to the substrate surface or remain suspended in the bulk solution, while those attached usually grow faster than those suspended due to their proximity to food resources. This imbalanced growth provides a plausible explanation to the inverse relationship between microbial concentration and their specific growth rate as conveyed in the Contois equation. Based on a theoretical derivation, this study revealed that the Contois equation holds true only when attached microbes substantially obstruct the access of food to their suspended counterparts. On the other hand, when plentiful insoluble substrate surfaces are exposed for cell attachment, the Contois equation will be reduced back to the classic Monod equation.     

Formation of bundles of microfilaments during spreading of fibroblasts on the substrate

Electron microscopy was used to study the sites of formation of bundles of parallel microfilaments in the early stages of spreading of normal mouse embryo fibroblasts on the substrate. Bundles of microfilaments were not found in suspended cells. Contact of the surface of spherical cells with the substrate was not sufficient for the formation of bundles: these bundles were not seen near the under surface of cells that were already attached to the substrate but had not yet developed cytoplasmic outgrowths at their periphery. Peripheral cytoplasmic outgrowths (microspikes and lamellar processes) attached to the substrate were found to be the only sites of localization of the first bundles of microfilaments seen in the spreading cells. It is suggested that surface and/or cytoplasm of the newly-formed peripheral cytoplasmic outgrowth may have some special properties necessary for the initiation of the development of microfilament bundles.     

Limitations of SLLVY-AMC in calpain and proteasome measurements

Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (SLLVY-AMC) is a fluorogenic substrate used to measure calpain activity and the “chymotrypsin-like” activity of the 20s proteasome. The goal of this study was to determine the relative role of calpains and the proteasome on SLLVY-AMC cleavage in attached and suspended renal epithelial cells (NRK-52E). The proteasome inhibitor epoxomicin did not inhibit purified calpain 1 or calpain 10 cleavage of SLLVY-AMC. Epoxomicin inhibited 11% of total SLLVY-AMC cleavage in attached cells and increasing concentrations of the calpain inhibitor calpeptin were additive. In contrast, cell suspensions had a 3.5-fold higher rate of SLLVY-AMC cleavage, epoxomicin inhibited cleavage 65% and calpeptin inhibited cleavage an additional 26%. Calpeptin alone also inhibited proteasomal activity. In conclusion, (1) SLLVY-AMC is cleaved in cells by calpain and the proteasome, (2) proteasome activity can be measured with epoxomicin, and (3) calpeptin can inhibit proteasome activity in some cases; thus limiting the use of SLLVY-AMC and calpeptin.     

The relation between surface area and anchorage dependence of growth in hamster and mouse fibroblasts.

Anchorage dependence can be defined as an increase in proliferation which is seen when cells are allowed to attach to a solid surface. We have measured this increase by time-lapse cinematography and other methods, and have compared it with measurements of the change in surface area which also occurs. Anchorage dependence can be varied over very wide limits in cells of the NIL-8 hamster fibroblast line by varying the concentration of serum in the medium. Thus in a concentration of 5% serum, the transition probability (a measure of growth rate) of attached cells is 8 times greater than in suspension. When the serum concentration is raised to 66%, attached and suspended cells grow at the same rate. We have found that this change in growth is accompanied by a corresponding change in the exposed surface area of the cells. Thus in 5% serum, the area of attached is 3 times greater than suspended cells, while in 66% serum, the areas of attached and suspended cells are equal. There is also a significant relationship between the area and the total amount of growth achieved, since both suspended colonies and attached confluent monolayers appear to stop growing at the same surface: volume ratio. Several aspects of this relationship between anchorage dependence and area are also seen when other different types of cell are cultured in the suspended and attached state, all at the same serum concentration. These experiments show that change in exposed surface area can provide a complete explanation for the anchorage dependence of NIL-8 cells and suggest the possibility that this might also be true of animal cells in general.     

Effects of surface substances of untransformed fibroblastic cells on the adhesion and proliferation of neighboring cells: a study using fixed confluent cell sheets

To study the effects of surface materials of cells on the behavior of other neighboring cells in a crowded culture, confluent sheets of rat 3Y1 fibroblasts were fixed and then 3Y1 cells were seeded on to them. Among confluent sheets unfixed, fixed with formalin and fixed with ethanol and an empty plastic dish surface, the substrate activity to permit cell adhesion was compared. After confluent 3Y1 cells (mainly composed of cells with a G1-DNA content) were reseeded with fresh medium on to these substrates, the capacity to initiate DNA synthesis per attached cell was also compared. The substrate activity of the ethanol-fixed cell sheet to permit cell adhesion was as high as that of the empty dish surface, whereas that of the unfixed cell sheet and that of the formalin-fixed cell sheet were low. When the ethanol-fixed cell sheet and the empty dish surface were coated with the ethanol extract of the unfixed cell sheet, the substrate activity diminished, indicating that during the fixation process with ethanol an adhesion-inhibitory factor (s) was removed. The capacity to initiate DNA synthesis of each cell that had completed adhesion and spreading on the cell sheets unfixed, fixed with formalin, and fixed with ethanol was lower compared to the cell that had adhered to the empty dish surface. We conclude that factors over the 3Y1 cell surface inhibit the overlapping cell adhesion and the proliferation of cells contacting each other, resulting in the ordered cell configuration in the confluent culture.     

Caspase sensitive gold nanoparticle for apoptosis imaging in live cells

We developed a new apoptosis imaging probe with gold nanoparticles (AuNPs). A near-infrared fluorescence dye was attached to AuNP surface through the bridge of peptide substrate (DEVD). The fluorescence was quenched in physiological conditions due to the quenching effect of AuNP, and the quenched fluorescence was recovered after the DEVD had been cleaved by caspase-3, the enzyme involved in apoptotic process. The adhesion of DEVD substrates on AuNP surface was accomplished by conjugation of the 3,4-dihydroxy phenylalanine (DOPA) groups which are adhesive to inorganic surface and rich in mussels. This surface modification with DEVD substrates by DOPA groups resulted in increased stability of AuNP in cytosol condition for hours. Moreover, the cleavage of substrate and the dequenching process are very fast, and the cells did not need to be fixed for imaging. Therefore, the real-time monitoring of caspase activity could be achieved in live cells, which enabled early detection of apoptosis compared to a conventional apoptosis kit such as Annexin V-FITC. Therefore, our apoptosis imaging has great potential as a simple, inexpensive, and efficient apoptosis imaging probe for biomedical applications.     

A new dimension in suspension fusion techniques with polyethylene glycol.

Polyethylene glycol (PEG) induces the hybridization of mammalian cells at a much higher frequency when the cells are attached to a substrate during treatment than when the cells are treated in suspension. Since many cell types, e.g., lymphocytes, cannot attach to a substrate, a new technique for the PEG-induced fusion of cells in suspension was developed. This technique, referred to as "pancake fusion," is based on the centrifugation of suspended cells onto a coverslip and the PEG treatment of the cells on the coverslip as if they were attached to a substrate. With this technique, the frequency of hybridization of human white blood cells, which are incapable of attaching to a substrate, can be greatly increased.     

Increased mass transfer to microorganisms with fluid motion

The effect of fluid flow and laminar shear on bacterial uptake was examined under conditions representative of the fluid environment of unattached and attached cells in wastewater treatment bioreactors. Laminar shear rates below 50 s(-1) did not increase leucine uptake by suspended cultures of Zoogloea ramigera. However, leucine uptake by cells fixed in a flow field of approximately 1 mm s(-1) was 55-65% greater than uptake by suspended cells. Enhanced microbial uptake with advective motion is consistent with mass transfer rates calculated using Sherwood number correlations. Advective flow increases microbial uptake by increasing collisions between substrate molecules and cells through compression of the concentration boundary layer surrounding a cell. The rate of leucine uptake suggests that binding proteins used to transport leucine into the cell can occupy approximately 1% of the cell surface area.     

Label-free reflectometric interference microchip biosensor based on nanoporous alumina for detection of circulating tumour cells

In this report, a label-free reflectometric interference spectroscopy (RIfS) based microchip biosensor for the detection of circulating tumour cells (CTCs) is demonstrated. Highly ordered nanoporous anodic aluminium oxide (AAO) fabricated by electrochemical anodization of aluminium foil was used as the RIfS sensing platform. Biotinylated anti-EpCAM antibody that specifically binds to human cancer cells of epithelial origin such as pancreatic cancer cells (PANC-1) was covalently attached to the AAO surface through multiple surface functionalization steps. Whole blood or phosphate buffer saline spiked with low numbers of pancreatic cancer cells were successfully detected by specially designed microfluidic device incorporating an AAO RIfS sensor, without labour intensive fluorescence labelling and/or pre-enhancement process. Our results show that the developed device is capable of selectively detecting of cancer cells, within a concentrations range of 1000-100,000 cells/mL, with a detection limit of <1000 cells/mL, a response time of <5 min and sample volume of 50 μL of. The presented RIfS method shows considerable promise for translation to a rapid and cost-effective point-of-care diagnostic device for the detection of CTCs in patients with metastatic cancer.     

Growth behaviors of bacteria in biofouling cake layer in a dead-end microfiltration system

The growth behaviors of three bacterial species, i.e. Escherichia coli, Pseudomonas putida and Aquabaculum hongkongensis, in biofouling cake layer (attached form) were investigated using an unstirred dead-end continuous microfiltration system, and were compared with those in suspended form. Results showed that all the three bacteria had larger average growth rates in suspended form than in attached form under high substrates levels. Under oligotrophic conditions, the average growth rates in the attached form were faster than those in the suspended form, especially for A. hongkongensis. The growth behaviors analysis presented the same results due to all the tested bacteria had higher maximum growth rate and saturation constant in suspended form than attached form, indicating the dominant growth mode would be shifted from attached form to suspended form with substrate concentration increase. Finally, total filtration resistance determined in the experiments increased significantly with the bacterial growth in filtration system.     

A TECHNIQUE FOR ULTRACRYOTOMY OF CELL SUSPENSIONS AND TISSUES

Ultracryotomy of fixed tissue has been investigated for a number of years but, so far, success has been limited for several reasons. The simple technique herein reported allows the ultracryotomy not only of a variety of tissues but also of single cells in suspension, with a preservation and visualization of ultrastructural detail at least equivalent to that obtained with conventional embedding procedures. In this technique, sucrose is infused into glutaraldehyde-fixed tissue pieces before freezing for the purpose of controlling the sectioning consistency. By choosing the proper combinations of sucrose concentration and sectioning temperature, a wide variety of tissues can be smoothly sectioned. Isolated cells, suspended in a sucrose solution, are sectioned by sectioning the frozen droplet of the suspension. A small liquid droplet of a saturated or near-saturated sucrose solution, suspended on the tip of an eyelash probe, is used to transfer frozen sections from the knife edge onto a grid substrate or a water surface. Upon melting of the sections on the surface of the sucrose droplet, they are spread flat and smooth due to surface tension. When the section of a suspension of single cells melts, individual sections of cells remain confined to the small area of the droplet surface. These devices make it possible to cut wide dry sections, and to avoid flotation on dimethyl sulfoxide solutions. With appropriate staining procedures, well-preserved ultrastructural detail can be observed. The technique is illustrated with a number of tissue preparations and with suspensions of erythrocytes and bacterial cells.     

Surface thermodynamics of leukocyte and platelet adhesion to polymer surfaces

Adhesion of leukocytes and platelets to solid substrates of different surface tensions and hence different wettability is studied from a thermodynamic point of view. A simple thermodynamic model predicts that cellular adhesion should increase with increasing surface tension of the solid substrate if the surface tension of the medium in which the cells are suspended is lower than the surface tension of the cells. If the surface tension of the suspending medium is higher than that of the cells, the opposite behavior is predicted. These predictions are borne out completely by neutrophil adhesion tests, where the surface tension of the aequeous suspending medium is varied by addition of dimethyl sulfoxide (DMSO). Platelet adhesion experiments also confirm these predictions, the only difference being that surface tensions of the suspending medium above that of the platelets cannot be realized, owing to exudation of surface active solutes from the platelets. Utilization of the thermodynamic prediction that cellular adhesion should become independent of the surface tension of the substrate when the surface tensions of the cells and that of the suspending medium are equal leads to a value of the surface tension of neutrophils of 69.0 erg/cm^2,† in excellent agreement with the value obtained from contact angles measured on layers of cells.     

Do brachiopods show substrate‐related phenotypic variation? A case study from the Burgess Shale

As sessile, benthic filter feeders, brachiopods share an intimate relationship with their chosen substrate. Individuals of Micromitra burgessensis in the Burgess Shale Formation are preserved in life position, attached to a range of hard substrates, including skeletal debris, conspecific brachiopods, sponges and enigmatic tubes. Here we investigate the phenotypic variability of M. burgessensis associated with differing substrate attachments. We apply geometric morphometrics to test for variation by plotting landmarks on the exterior of ventral and dorsal valves of M. burgessensis specimens that are preserved attached to different substrates. Using principal component, canonical variate analyses and anova , we determine that there is some variation in shape related to substrate. Canonical variate analyses, for ventral valves and dorsal valves, indicate that specimens attached to the same substrate are recognizable in shape from specimens attached to other substrate types. The strength of differentiation however, is not robust and combined with our discriminate analysis of separate populations suggests that there is the potential for substrates to exercise only weak control over the morphology of Brachiopoda.     

A comparison of the growth and starvation responses of Acanthamoeba castellanii and Hartmannella vermiformis in the presence of suspended and attached Escherichia coli K12

The growth and starvation responses of Acanthamoeba castellanii and Hartmannella vermiformis were investigated in the presence and absence of Escherichia coli on an agar surface or within shaken suspensions. The amoebae perceived all the suspended systems to be unfavourable for growth, despite being challenged with high levels of prey, and as a consequence they exhibited a starvation response. However, the response differed between species, with A. castellanii producing characteristic cysts and H. vermiformis producing round bodies. These amoebic forms were reactivated into feeding trophozoites in the presence of bacterial aggregates, which formed in the suspended systems after 68 h of incubation. In contrast, both species of amoebae grew well in the presence of attached E. coli at a concentration of 1 x 10(6) cells cm(-2) of agar and yielded specific growth rates of c. 0.04 h(-1). Starvation responses were induced at the end of the growth phase, and these were equivalent to those recorded in the suspended systems. We conclude that, when suspended, amoebae in the 'floating form' cannot feed effectively on suspended prey, and hence the starvation response is initiated. Thus the majority of amoebic feeding is via trophozoite grazing of attached bacterial prey.     

Surface thermodynamics of leukocyte and platelet adhesion to polymer surfaces.

Adhesion of leukocytes and platelets to solid substrates of different surface tensions and hence different wettability is studied from a thermodynamic point of view. A simple thermodynamic model predicts that a cellular adhesion should increase with increasing surface tension of the solid substrate if the surface tension of the medium in which the cells are suspended is lower than the surface tension of the cells. If the surface tension of the suspending medium is higher than that of the cells, the opposite behavior is predicted. These predictions are borne out completely by neutrophil adhesion tests, where the surface tension of the aqueous suspending medium is varied by addition of dimethyl sulfoxide (DMSO). Platelet adhesion experiments also confirm these predictions, the only difference being that surface tensions of the suspending medium above that of the platelets cannot be realized, owing to exudation of surface active solutes from the platelets. Utilization of the thermodynamic prediction that cellular adhesion should become independent of the surface tension of the substrate when the surface tensions of the cells and that of the suspending medium are equal leads to a value of the surface tension of neutrophils of 69.0 erg/cm(2), in excellent agreement with the value obtained from contact angles measured on layers of cells.     

Expression of capacitation-dependent changes in chlortetracycline fluorescence patterns in mouse spermatozoa requires a suitable glycolysable substrate

Chlortetracycline (CTC) fluorescence patterns were assessed in epididymal mouse sperm suspensions capacitated in exogenous substrate-containing and substrate-free media. A capacitation-dependent transition from a majority of acrosome-intact cells expressing the uncapacitated F pattern of fluorescence to a majority with the capacitated acrosome-intact B and acrosome-reacted AR patterns was confirmed for suspensions incubated a total of 120 min in the presence of a glycolysable substrate, glucose. In contrast, assessment of spermatozoa incubated for 120 min in substrate-free medium revealed a majority of cells with the uncapacitated F pattern, despite an earlier demonstration that such cells are essentially capacitated: upon the introduction of glucose, suspensions are immediately highly fertile. When a suitable glycolysable substrate, either glucose or mannose but not fructose, was added to such suspensions, the distribution of CTC patterns changed within 10 min to a majority of B and AR patterns. Furthermore, the degree of change from uncapacitated to capacitated patterns was substrate concentration-dependent. In contrast, the introduction of the non-metabolizable substrates 2-deoxyglucose and 3-0-methylglucose and the oxidizable substrates sodium pyruvate and sodium lactate caused no change in the patterns from those seen in substrate-free medium. The in-vitro fertilizing ability of sperm suspensions to which increasing amounts of glucose or mannose were added, after initial substrate-free preincubation, directly paralleled the changes in CTC patterns and was as rapid as for suspensions incubated continuously in either hexose. We therefore conclude that the alteration in position of surface components to which CTC binds is not only capacitation-dependent, but also energy-dependent. In the absence of an appropriate exogenous glycolysable substrate, the final transition cannot occur, even though the cells are essentially capacitated.     

Do β-lactam antibiotics permeabilize the outer membrane of gram-negative bacteria? An electrochemical investigation

Abstract The extent of short-term adhesion of various suspension-cultured plant cell species to polymer substrates exhibiting a wide range of surface tensions was examined. Adhesion of cells with a relatively low surface tension, suspended in distilled water, to the polymers fluorinated ethylenepropylene (FEP), polystyrene (PS), polyethylene terephthalate (PET), and sulphonated polystyrene (SPS) increased with decreasing substrate surface tension following the sequence SPS < PET < PS < FEP. These results are in agreement with the predictions of a thermodynamic model of particle adhesion which considers the role of the substrate, suspending-liquid, and cellular surface tensions. In contrast, little adhesion of relatively high surface tension cells to any of the polymer substrates was observed. Electrostatic repulsive forces between these cells and the polymer surface prevent adhesion because the magnitude of the attractive van der Waals force is small. A correlation was observed between the general adhesiveness of the various cultured plant cell species, especially to the low surface tension substrates, and the cellular surface tension determined by measuring the water contact angle on smooth layers of the cells. The cellular surface tensions ranged from approximately 42 mJ/m² for Digitalis purpurea cells to approximately 70mJ/m² for Papaver somniferum cells. Adhesion of cells to the polymer substrates increased with decreasing cellular surface tension under otherwise identical conditions. These results are also consistent with thermodynamic model predictions.