Development and characterization of an organotypic model of Barrett's esophagus

Understanding the molecular and cellular processes underlying the development, maintenance, and progression of Barrett's esophagus (BE) presents an empirical challenge because there are no simple animal models and standard 2D cell culture can distort cellular processes. Here we describe a three-dimensional (3D) cell culture system to study BE. BE cell lines (CP-A, CP-B, CP-C, and CP-D) and esophageal squamous keratinocytes (EPC2) were cultured on a matrix consisting of esophageal fibroblasts and collagen. Comparison of growth and cytokeratin expression in the presence of all-trans retinoic acid or hydrochloric acid was made by immunohistochemistry and Alcian Blue staining to determine which treatments produced a BE phenotype of columnar cytokeratin expression in 3D culture. All-trans retinoic acid differentially affected the growth of BE cell lines in 3D culture. Notably, the non-dyplastic metaplasia-derived cell line (CP-A) expressed reduced squamous cytokeratins and enhanced columnar cytokeratins upon ATRA treatment. ATRA altered the EPC2 squamous cytokeratin profile towards a more columnar expression pattern. Cell lines derived from patients with high-grade dysplasia already expressed columnar cytokeratins and therefore did not show a systematic shift toward a more columnar phenotype with ATRA treatment. ATRA treatment, however, did reduce the squamoid-like multilayer stratification observed in all cell lines. As the first study to demonstrate long-term 3D growth of BE cell lines, we have determined that BE cells can be cultured for at least 3 weeks on a fibroblast/collagen matrix and that the use of ATRA causes a general reduction in squamous-like multilayered growth and an increase in columnar phenotype with the specific effects cell-line dependent.     

Atomic force microscope elastography reveals phenotypic differences in alveolar cell stiffness.

To understand the connection between alveolar mechanics and key biochemical events such as surfactant secretion, one first needs to characterize the underlying mechanical properties of the lung parenchyma and its cellular constituents. In this study, the mechanics of three major cell types from the neonatal rat lung were studied; primary alveolar type I (AT1) and type II (AT2) epithelial cells and lung fibroblasts were isolated using enzymatic digestion. Atomic force microscopy indentation was used to map the three-dimensional distribution of apparent depth-dependent pointwise elastic modulus. Histograms of apparent modulus data from all three cell types indicated non-Gaussian distributions that were highly skewed and appeared multimodal for AT2 cells and fibroblasts. Nuclear stiffness in all three cell types was similar (2.5+/-1.0 kPa in AT1 vs. 3.1+/-1.5 kPa in AT2 vs. 3.3+/-0.8 kPa in fibroblasts; n=10 each), whereas cytoplasmic moduli were significantly higher in fibroblasts and AT2 cells (6.0+/-2.3 and 4.7+/-2.9 kPa vs. 2.5+/-1.2 kPa). In both epithelial cell types, actin was arranged in sparse clusters, whereas prominent actin stress fibers were observed in lung fibroblasts. No systematic difference in actin or microtubule organization was noted between AT1 and AT2 cells. Atomic force microscope elastography, combined with live-cell fluorescence imaging, revealed that the stiffer measurements in AT2 cells often colocalized with lamellar bodies. These findings partially explain reported heterogeneity of alveolar cell deformation during in situ lung inflation and provide needed data for better understanding of how mechanical stretch influences surfactant release.     

Mapping elasticity moduli of atherosclerotic plaque in situ via atomic force microscopy

Several studies have suggested that evolving mechanical stresses and strains drive atherosclerotic plaque development and vulnerability. Especially, stress distribution in the plaque fibrous capsule is an important determinant for the risk of vulnerable plaque rupture. Knowledge of the stiffness of atherosclerotic plaque components is therefore of critical importance. In this work, force mapping experiments using atomic force microscopy (AFM) were conducted in apolipoprotein E-deficient (ApoE(-/-)) mouse, which represents the most widely used experimental model for studying mechanisms underlying the development of atherosclerotic lesions. To obtain the elastic material properties of fibrous caps and lipidic cores of atherosclerotic plaques, serial cross-sections of aortic arch lesions were probed at different sites. Atherosclerotic plaque sub-structures were subdivided into cellular fibrotic, hypocellular fibrotic and lipidic rich areas according to histological staining. Hertz's contact mechanics were used to determine elasticity (Young's) moduli that were related to the underlying histological plaque structure. Cellular fibrotic regions exhibit a mean Young modulus of 10.4±5.7kPa. Hypocellular fibrous caps were almost six-times stiffer, with average modulus value of 59.4±47.4kPa, locally rising up to ～250kPa. Lipid rich areas exhibit a rather large range of Young's moduli, with average value of 5.5±3.5kPa. Such precise quantification of plaque stiffness heterogeneity will allow investigators to have prospectively a better monitoring of atherosclerotic disease evolution, including arterial wall remodeling and plaque rupture, in response to mechanical constraints imposed by vascular shear stress and blood pressure.     

Mechano-acoustic determination of Young's modulus of articular cartilage

The compressive stiffness of an elastic material is traditionally characterized by its Young's modulus. Young's modulus of articular cartilage can be directly measured using unconfined compression geometry by assuming the cartilage to be homogeneous and isotropic. In isotropic materials, Young's modulus can also be determined acoustically by the measurement of sound speed and density of the material. In the present study, acoustic and mechanical techniques, feasible for in vivo measurements, were investigated to quantify the static and dynamic compressive stiffness of bovine articular cartilage in situ. Ultrasound reflection from the cartilage surface, as well as the dynamic modulus were determined with the recently developed ultrasound indentation instrument and compared with the reference mechanical and ultrasound speed measurements in unconfined compression (n=72). In addition, the applicability of manual creep measurements with the ultrasound indentation instrument was evaluated both experimentally and numerically. Our experimental results indicated that the sound speed could predict 47% and 53% of the variation in the Young's modulus and dynamic modulus of cartilage, respectively. The dynamic modulus, as determined manually with the ultrasound indentation instrument, showed significant linear correlations with the reference Young's modulus (r(2)=0.445, p<0.01, n=70) and dynamic modulus (r(2)=0.779, p<0.01, n=70) of the cartilage. Numerical analyses indicated that the creep measurements, conducted manually with the ultrasound indentation instrument, were sensitive to changes in Young's modulus and permeability of the tissue, and were significantly influenced by the tissue thickness. We conclude that acoustic parameters, i.e. ultrasound speed and reflection, are indicative to the intrinsic mechanical properties of the articular cartilage. Ultrasound indentation instrument, when further developed, provides an applicable tool for the in vivo detection of cartilage mechano-acoustic properties. These techniques could promote the diagnostics of osteoarthrosis.     

Static versus dynamic gerbil tympanic membrane elasticity: derivation of the complex modulus

An accurate estimation of tympanic membrane stiffness is important for realistic modelling of middle ear mechanics. Tympanic membrane stiffness has been investigated extensively under either quasi-static or dynamic loading conditions. It is known that biological tissues are sensitive to strain rate. Therefore, in this work, the mechanical behaviour of the tympanic membrane was studied under both quasi-static and dynamic loading conditions. Experiments were performed on the pars tensa of four gerbil tympanic membranes. A custom-built indentation apparatus was used to perform in situ tissue indentations and testing was done applying both quasi-static and dynamic sinusoidal indentations up to 8.2?Hz. The unloaded shape of the tympanic membrane was measured and used to create specimen-specific finite element models to simulate the experiments. The frequency dependent Young's modulus of each specimen was then estimated by an inverse analysis in which the error between experimental and simulated indentation data was optimised for each indentation frequency separately. Using an 8?μm central region thickness, we found Young's moduli between 71 and 106?MPa (n = 4) at 0.2?Hz indentation frequency. A standard linear viscoelastic model and a viscoelastic model with a continuous relaxation spectrum were used to derive a complex modulus in the frequency domain. Due to experimental limitations, the indentation frequency upper limit was 8.2?Hz. The average relative modulus increase in this domain was 14% and the increase was the strongest below 6?Hz.     

Investigating the effect of cell substrate on cancer cell stiffness by optical tweezers

The mechanical properties of cells are influenced by their microenvironment. Here we report cell stiffness alteration by changing the cell substrate stiffness for isolated cells and cells in contact with other cells. Polydimethylsiloxane (PDMS) is used to prepare soft substrates with three different stiffness values (173, 88 and 17 kPa respectively). Breast cancer cells lines, namely HBL-100, MCF-7 and MDA-MB-231 with different level of aggressiveness are cultured on these substrates and their local elasticity is investigated by vertical indentation of the cell membrane. Our preliminary results show an unforeseen behavior of the MDA-MB-231 cells. When cultured on glass substrate as isolated cells, they are less stiff than the other two types of cells, in agreement with the general statement that more aggressive and metastatic cells are softer. However, when connected to other cells the stiffness of MDA-MB-231 cells becomes similar to the other two cell lines. Moreover, the stiffness of MDA-MB-231 cells cultured on soft PDMS substrates is significantly higher than the stiffness of the other cell types, demonstrating thus the strong influence of the environmental conditions on the mechanical properties of the cells.     

A biphasic model for micro-indentation of a hydrogel-based contact lens

The stiffness and hydraulic permeability of soft contact lenses may influence its clinical performance, e.g., on-eye movement, fitting, and wettability, and may be related to the occurrence of complications; e.g., lesions. It is therefore important to determine these properties in the design of comfortable contact lenses. Micro-indentation provides a nondestructive means of measuring mechanical properties of soft, hydrated contact lenses. However, certain geometrical and material considerations must be taken into account when analyzing output force-displacement (F-D) data. Rather than solely having a solid response, mechanical behavior of hydrogel contact lenses can be described as the coupled interaction between fluid transport through pores and solid matrix deformation. In addition, indentation of thin membranes ( approximately 100 microm) requires special consideration of boundary conditions at lens surfaces and at the indenter contact region. In this study, a biphasic finite element model was developed to simulate the micro-indentation of a hydrogel contact lens. The model accounts for a curved, thin hydrogel membrane supported on an impermeable mold. A time-varying boundary condition was implemented to model the contact interface between the impermeable spherical indenter and the lens. Parametric studies varying the indentation velocities and hydraulic permeability show F-D curves have a sensitive region outside of which the force response reaches asymptotic limits governed by either the solid matrix (slow indentation velocity, large permeability) or the fluid transport (high indentation velocity, low permeability). Using these results, biphasic properties (Young's modulus and hydraulic permeability) were estimated by fitting model results to F-D curves obtained at multiple indentation velocities (1.2 and 20 microm/s). Fitting to micro-indentation tests of Etafilcon A resulted in an estimated permeability range of 1.0 x 10(-15) to 5.0 x 10(-15) m(4)N s and Young's modulus range of 130 to 170 kPa.     

Elasticity of normal and cancerous human bladder cells studied by scanning force microscopy

Scanning force microscopy was used for the determination of the elastic properties of living cells in their culture conditions. The studies were carried out on human epithelial cells. Two similar lines of normal cells (Hu609 and HCV29) and three cancerous ones (Hu456, T24, BC3726) were measured using the scanning force microscope in order to collect the force versus indentation curves. The BC3726 line originates from the HCV29 cell line which was transformed by the v-ras oncogene. To evaluate their elastic properties, Young's modulus values were determined. The present study has shown that normal cells have a Young's modulus of about one order of magnitude higher than cancerous ones. Such a change might be attributed to a difference in the organisation of cell cytoskeletons and requires further studies. Received: 30 April 1998 / Revised version: 17 December 1998 / Accepted: 21 January 1999     

Alterations in the Young's modulus and volumetric properties of chondrocytes isolated from normal and osteoarthritic human cartilage

The mechanical environment of the chondrocyte is an important factor that influences the maintenance of the articular cartilage extracellular matrix. Previous studies have utilized theoretical models of chondrocytes within articular cartilage to predict the stress-strain and fluid flow environments around the cell, but little is currently known regarding the cellular properties which are required for implementation of these models. The objectives of this study were to characterize the mechanical behavior of primary human chondrocytes and to determine the Young's modulus of chondrocytes from non-osteoarthritic ('normal') and osteoarthritic cartilage. A second goal was to quantify changes in the volume of isolated chondrocytes in response to mechanical deformation. The micropipette aspiration technique was used to measure the deformation of a single chondrocyte into a glass micropipette in response to a prescribed pressure. The results of this study indicate that the human chondrocyte behaves as a viscoelastic solid. No differences were found between the Young's moduli of normal (0.65+/-0.63 kPa, n = 44) and osteoarthritic chondrocytes (0.67+/-0.86 kPa, n = 69, p = 0.93). A significant difference in cell volume was observed immediately and 600 s after complete aspiration of the cell into the pipette (p < 0.001), and the magnitude of this volume change between normal (11+/-11%, n = 40) and osteoarthritic (20+/-11%, n = 41) chondroctyes was significantly different at both time points (p < 0.002). This finding suggests that chondrocytes from osteoarthritic cartilage may have altered volume regulation capabilities in response to mechanical deformation. The mechanical and volumetric properties determined in this study will be of use in analytical and finite element models of chondrocyte-matrix interactions in order to better predict the mechanical environment of the cell in vivo.     

基于原子力显微镜测量内耳螺旋器的弹性特征

应用原子力显微镜分析内耳螺旋器(Corti器)不同部位的弹性特征。采用豚鼠内耳基底膜底回新鲜标本,用原子力显微镜在液相接触式测量,获得不同部位力曲线。经计算,对应Corti器相当于Hensen细胞、外毛细胞、柱细胞、内毛细胞、内指细胞、盖膜的部位及基底膜底面局部,其杨氏模量均值分别为46±1.7、59±0.9、250±31、140±2.8、430±29.9、210±7.2和230±8.8 kPa。结果表明,基底膜径向排列的组织结构不同,杨氏模量存在明显差异,在整块基底膜标本上测量Corti器各结构的杨氏模量能更准确地反映它们在生理状态下的弹性特征。     

Measuring the viscoelastic properties of human platelets with the atomic force microscope.

We have measured force curves as a function of the lateral position on top of human platelets with the atomic force microscope. These force curves show the indentation of the cell as the tip loads the sample. By analyzing these force curves we were able to determine the elastic modulus of the platelet with a lateral resolution of approximately 100 nm. The elastic moduli were in a range of 1-50 kPa measured in the frequency range of 1-50 Hz. Loading forces could be controlled with a resolution of 80 pN and indentations of the platelet could be determined with a resolution of 20 nm.     

Depth-sensing analysis of cytoskeleton organization based on AFM data

Atomic force microscopy is a common technique used to determine the elastic properties of living cells. It furnishes the relative Young’s modulus, which is typically determined for indentation depths within the range 300–500 nm. Here, we present the results of depth-sensing analysis of the mechanical properties of living fibroblasts measured under physiological conditions. Distributions of the Young’s moduli were obtained for all studied cells and for every cell. The results show that for small indentation depths, histograms of the relative values of the Young’s modulus described the regions rich in the network of actin filaments. For large indentation depths, the overall stiffness of a whole cell was obtained, which was accompanied by a decrease of the modulus value. In conclusion, the results enable us to describe the non-homogeneity of the cell cytoskeleton, particularly, its contribution linked to actin filaments located beneath the cell membrane. Preliminary results showing a potential application to improve the detection of cancerous cells, have been presented for melanoma cell lines.     

Warburg and Crabtree Effects in Premalignant Barrett's Esophagus Cell Lines with Active Mitochondria

Background

Increased glycolysis is a hallmark of cancer metabolism, yet relatively little is known about this phenotype at premalignant stages of progression. Periodic ischemia occurs in the premalignant condition Barrett''s esophagus (BE) due to tissue damage from chronic acid-bile reflux and may select for early adaptations to hypoxia, including upregulation of glycolysis.

Methodology/Principal Findings

We compared rates of glycolysis and oxidative phosphorylation in four cell lines derived from patients with BE (CP-A, CP-B, CP-C and CP-D) in response to metabolic inhibitors and changes in glucose concentration. We report that cell lines derived from patients with more advanced genetically unstable BE have up to two-fold higher glycolysis compared to a cell line derived from a patient with early genetically stable BE; however, all cell lines preserve active mitochondria. In response to the glycolytic inhibitor 2-deoxyglucose, the most glycolytic cell lines (CP-C and CP-D) had the greatest suppression of extra-cellular acidification, but were able to compensate with upregulation of oxidative phosphorylation. In addition, these cell lines showed the lowest compensatory increases in glycolysis in response to mitochondrial uncoupling by 2,4-dinitrophenol. Finally, these cell lines also upregulated their oxidative phosphorylation in response to glucose via the Crabtree effect, and demonstrate a greater range of modulation of oxygen consumption.

Conclusions/Significance

Our findings suggest that cells from premalignant Barrett''s esophagus tissue may adapt to an ever-changing selective microenvironment through changes in energy metabolic pathways typically associated with cancer cells.     

Simple display system of mechanical properties of cells and their dispersion

The mechanical properties of cells are unique indicators of their states and functions. Though, it is difficult to recognize the degrees of mechanical properties, due to small size of the cell and broad distribution of the mechanical properties. Here, we developed a simple virtual reality system for presenting the mechanical properties of cells and their dispersion using a haptic device and a PC. This system simulates atomic force microscopy (AFM) nanoindentation experiments for floating cells in virtual environments. An operator can virtually position the AFM spherical probe over a round cell with the haptic handle on the PC monitor and feel the force interaction. The Young's modulus of mesenchymal stem cells and HEK293 cells in the floating state was measured by AFM. The distribution of the Young's modulus of these cells was broad, and the distribution complied with a log-normal pattern. To represent the mechanical properties together with the cell variance, we used log-normal distribution-dependent random number determined by the mode and variance values of the Young's modulus of these cells. The represented Young's modulus was determined for each touching event of the probe surface and the cell object, and the haptic device-generating force was calculated using a Hertz model corresponding to the indentation depth and the fixed Young's modulus value. Using this system, we can feel the mechanical properties and their dispersion in each cell type in real time. This system will help us not only recognize the degrees of mechanical properties of diverse cells but also share them with others.     

IL-1beta decreases the elastic modulus of human tenocytes.

Cellular responses to mechanical stimuli are regulated by interactions with the extracellular matrix, which, in turn, are strongly influenced by the degree of cell stiffness (Young's modulus). It was hypothesized that a more elastic cell could better withstand the rigors of remodeling and mechanical loading. It was further hypothesized that interleukin-1beta (IL-1beta) would modulate intracellular cytoskeleton polymerization and regulate cell stiffness. The purpose of this study was to investigate the utility of IL-1beta to alter the Young's modulus of human tenocytes. Young's modulus is the ratio of the stress to the strain, E = stress/strain = (F/A)/(deltaL/L0), where L0 is the equilibrium length, deltaL is the length change under the applied stress, F is the force applied, and A is the area over which the force is applied. Human tenocytes were incubated with 100 pM recombinant human IL-1beta for 5 days. The Young's modulus was reduced by 27-63%. Actin filaments were disrupted in >75% of IL-1beta-treated cells, resulting in a stellate shape. In contrast, immunostaining of alpha-tubulin showed increased intensity in IL-1beta-treated tenocytes. Human tenocytes in IL-1beta-treated bioartificial tendons were more tolerant to mechanical loading than were untreated counterparts. These results indicate that IL-1beta reduced the Young's modulus of human tenocytes by disrupting the cytoskeleton and/or downregulating the expression of actin and upregulating the expression of tubulins. The reduction in cell modulus may help cells to survive excessive mechanical loading that may occur in damaged or healing tendons.     

Non-Hertzian approach to analyzing mechanical properties of endothelial cells probed by atomic force microscopy

Detailed measurements of cell material properties are required for understanding how cells respond to their mechanical environment. Atomic force microscopy (AFM) is an increasingly popular measurement technique that uniquely combines subcellular mechanical testing with high-resolution imaging. However, the standard method of analyzing AFM indentation data is based on a simplified "Hertz" theory that requires unrealistic assumptions about cell indentation experiments. The objective of this study was to utilize an alternative "pointwise modulus" approach, that relaxes several of these assumptions, to examine subcellular mechanics of cultured human aortic endothelial cells (HAECs). Data from indentations in 2- to 5-microm square regions of cytoplasm reveal at least two mechanically distinct populations of cellular material. Indentations colocalized with prominent linear structures in AFM images exhibited depth-dependent variation of the apparent pointwise elastic modulus that was not observed at adjacent locations devoid of such structures. The average pointwise modulus at an arbitrary indentation depth of 200 nm was 5.6+/-3.5 kPa and 1.5+/-0.76 kPa (mean+/-SD, n=7) for these two material populations, respectively. The linear structures in AFM images were identified by fluorescence microscopy as bundles of f-actin, or stress fibers. After treatment with 4 microM cytochalasin B, HAECs behaved like a homogeneous linear elastic material with an apparent modulus of 0.89+/-0.46 kPa. These findings reveal complex mechanical behavior specifically associated with actin stress fibers that is not accurately described using the standard Hertz analysis, and may impact how HAECs interact with their mechanical environment.     

Creep indentation of single cells

An apparatus for creep indentation of individual adherent cells was designed, developed, and experimentally validated. The creep cytoindentation apparatus (CCA) can perform stress-controlled experiments and measure the corresponding deformation of single anchorage-dependent cells. The apparatus can resolve forces on the order of 1 nN and cellular deformations on the order of 0.1 micron. Experiments were conducted on bovine articular chondrocytes using loads on the order of 10 nN. The experimentally observed viscoelastic behavior of these cells was modeled using the punch problem and standard linear solid. The punch problem yielded a Young's modulus of 1.11 +/- 0.48 kPa. The standard linear solid model yielded an instantaneous elastic modulus of 8.00 +/- 4.41 kPa, a relaxed modulus of 1.09 +/- 0.54 kPa, an apparent viscosity of 1.50 +/- 0.92 kPa-s, and a time constant of 1.32 +/- 0.65 s. To our knowledge, this is the first time that stress-controlled indentation testing has been applied at the single cell level. This methodology represents a new tool in understanding the mechanical nature of anchorage-dependent cells and mechanotransductional pathways.     

Elasticity of native and cross-linked polyelectrolyte multilayer films

Mechanical properties of polyelectrolyte multilayer films were studied by nanoindentation using the atomic force microscope (AFM). Force-distance measurements using colloidal probe tips were systematically obtained for supported films of poly(L-lysine) and hyaluronan that are suited to bio-application. Both native and covalently cross-linked films were studied as a function of increasing layer number, which increases film thickness. The effective Young's modulus perpendicular to the film, Eperpendicular, was determined to be a function of film thickness, cross-linking, and sample age. Thick PEM films exhibited a lower Eperpendicular than thinner PEM, whereas the Young's modulus of cross-linked films was more than 10-fold larger than native films. Moduli range from approximately 20 kPa for native films up to approximately 800 kPa for cross-linked ones. Young's moduli increased slightly with sample age, plateauing after approximately 4 weeks. Spreading of smooth muscle cells on these substrates with pre-attached collagen proved to be highly dependent on film rigidity with stiffer films giving greater cell spreading.     

Probing elasticity and adhesion of live cells by atomic force microscopy indentation

Atomic force microscopy (AFM) indentation has become an important technique for quantifying the mechanical properties of live cells at nanoscale. However, determination of cell elasticity modulus from the force–displacement curves measured in the AFM indentations is not a trivial task. The present work shows that these force–displacement curves are affected by indenter-cell adhesion force, while the use of an appropriate indentation model may provide information on the cell elasticity and the work of adhesion of the cell membrane to the surface of the AFM probes. A recently proposed indentation model (Sirghi, Rossi in Appl Phys Lett 89:243118, 2006), which accounts for the effect of the adhesion force in nanoscale indentation, is applied to the AFM indentation experiments performed on live cells with pyramidal indenters. The model considers that the indentation force equilibrates the elastic force of the cell cytoskeleton and the adhesion force of the cell membrane. It is assumed that the indenter-cell contact area and the adhesion force decrease continuously during the unloading part of the indentation (peeling model). Force–displacement curves measured in indentation experiments performed with silicon nitride AFM probes with pyramidal tips on live cells (mouse fibroblast Balb/c3T3 clone A31-1-1) in physiological medium at 37°C agree well with the theoretical prediction and are used to determine the cell elasticity modulus and indenter-cell work of adhesion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stiffness Tomography by Atomic Force Microscopy

The atomic force microscope is a convenient tool to probe living samples at the nanometric scale. Among its numerous capabilities, the instrument can be operated as a nano-indenter to gather information about the mechanical properties of the sample. In this operating mode, the deformation of the cantilever is displayed as a function of the indentation depth of the tip into the sample. Fitting this curve with different theoretical models permits us to estimate the Young's modulus of the sample at the indentation spot. We describe what to our knowledge is a new technique to process these curves to distinguish structures of different stiffness buried into the bulk of the sample. The working principle of this new imaging technique has been verified by finite element models and successfully applied to living cells.