Micropipette Aspiration Technique Research Articles

Determining β2-Integrin and Intercellular Adhesion Molecule 1 Binding Kinetics in Tumor Cell Adhesion to Leukocytes and Endothelial Cells by a Gas-driven Micropipette Assay

Interactions between polymorphonuclear neutrophils (PMNs) and tumor cells have been reported to facilitate the adhesion and subsequent extravasation of tumor cells through the endothelium under blood flow, both of which are mediated by binding β(2)-integrin to intercellular adhesion molecule 1 (ICAM-1). Here the adhesions between human WM9 metastatic melanoma cells, PMNs, and human pulmonary microvascular endothelial cells (HPMECs) were quantified by a gas-driven micropipette aspiration technique (GDMAT). Our data indicated that the cellular binding affinity of PMN-WM9 pair was 3.9-fold higher than that of the PMN-HPMEC pair. However, the effective binding affinities per molecular pair were comparable between the two cell pairs no matter whether WM9 cells or HPMECs were quiescent or cytokine-activated, indicating that the stronger adhesion between PMN-WM9 pair is mainly attributed to the high expression of ICAM-1 on WM9 cells. These results proposed an alternative mechanism, where WM9 melanoma cells adhere first with PMNs near vessel-wall regions and then bind to endothelial cells via PMNs under blood flow. In contrast, the adhesions between human MDA-MB-231 metastatic breast carcinoma cells and PMNs showed a comparable cellular binding affinity to PMN-HPMEC pair because the ICAM-1 expressions on MDA-MB-231 cells and HPMECs are similar. Furthermore, differences were observed in the intrinsic forward and reverse rates of the β(2)-integrin-ICAM-1 bond between PMN-TC and PMN-EC pairs. This GDMAT assay enables us to quantify the binding kinetics of cell adhesion molecules physiologically expressed on nucleated cells. The findings also further the understanding of leukocyte-facilitated tumor cell adhesion from the viewpoint of molecular binding kinetics.

Viscoelastic properties of human normal nucleus pulposus cells

Objective To study the viscoelastic properties of nucleus pulposus (NP) cells from human in vitro. Methods NP was obtained from discarded NP tissue of 3 scoliosis patients aged from 13 to 16 years. Pancreatin and collagenase type Ⅱ were used to digest NP and cells were isolated from NP. Type Ⅱ collagen immunofluorescence and Fan seaing were used to identify NP cells. The micropipette aspiration test was used in combination with a three-parameter viscoelastic solid model to measure the mechanical properties of NP cells. Results The mean diameter of the digested NP cells was ( 15.40 ± 1.83) μm. In response to a prescribed pressure, the NP cells exhibited viscolastic solid creep behavior, which was characterized initially by a jump in displacement followed by a monotony decreasing rate of deformation that generally reached an equilibrium. NP cells were deformed to a length as much as 2 times the radius of the micropipette without completely entering the micropipette. The viscolastic parameters were k1 (0. 101 ±0. 052) kPa, k2 (0. 353 ± 0. 199) kPa, and μ ( 3. 034 ± 1. 843 ) kPa· s, respectively. Only the k1 was positively correlated to the cell diameter (r =-0. 389, P ＜ 0. 05 ). Conclusion Human normal NP cells behave as a typical viscolastic solid creep. Micropipette aspiration technique is a valid method for the study on biomechanics of NP cells. Key words: Nucleus pulposus cells; Biomechanics; Viscoelastic properties

Inhibition of Intercellular Adhesion in Herpex Simplex Virus Infection by Glycyrrhizin

Herpes simplex virus (HSV) is one of the most common viruses infecting humans and animals. Cellular adhesion is increased in HSV and plays a role in pathogenesis of inflammatory response during this viral infection. In our study, we studied a potential role of glycyrrhizin in disrupting cellular adhesion in HSV. We isolated rat cerebral capillary vessel endothelial cells (CCECs) and polymorphonuclear leukocytes (PMN) and evaluated intercellular adhesion between these cells by micropipette aspiration technique. The adhesion force and stress between CCEC and PMN were significantly (P < 0.01) increased in HSV infection. Glycyrrhizin perfusion significantly (P < 0.01) reduced adhesion force and stress between CCEC and PMN. In conclusion, glycyrrhizin may attenuate inflammatory responses in HSV by inhibition of adhesion between CCEC and PMN.

Surface Protrusion of Human Umbilical Vein Endothelial Cells

During leukocyte rolling on the endothelium, membrane tethers can be extracted simultaneously from both leukocytes and endothelial cells. Tether extraction, which has been shown to stabilize leukocyte rolling, is preceded by surface protrusion, which dictates whether tether extraction can occur. Although surface protrusion of leukocytes has been characterized, surface protrusion of endothelial cells has not. In this work, we present a detailed study of surface protrusion of human umbilical vein endothelial cells (HUVECs). Using the micropipette aspiration technique, we measured the protrusional stiffness and the crossover force during HUVEC surface protrusion. We found that, compared with leukocytes, the protrusional stiffness and the crossover force of HUVECs were both larger at similar force loading rates. The values of these two parameters depended on temperature, the cytoskeletal integrity, α-actinin1, and whether CD31 or CD29 was used as the force handle. However, they did not depend on cell attachment state or intracellular calcium. These results show that similar mechanisms govern surface protrusion, hence also tether extraction in leukocytes and endothelial cells. They will help us understand and eventually control this critical step of the immune response.

Squeezing and Detachment of Living Cells

The interaction of living cells with their environment is linked to their adhesive and elastic properties. Even if the mechanics of simple lipid membranes is fairly well understood, the analysis of single cell experiments remains challenging in part because of the mechanosensory response of cells to their environment. To study the mechanical properties of living cells we have developed a tool that borrows from micropipette aspiration techniques, atomic force microscopy, and the classical Johnson-Kendall-Roberts test. We show results from a study of the adhesion properties of living cells, as well as the elastic response and relaxation. We present models that are applied throughout the different stages of an experiment, which indicate that the contribution of the different components of the cell are active at various stages of compression, retraction, and detachment. Finally, we present a model that attempts to elucidate the surprising logarithmic relaxation observed when the cell is subjected to a given deformation.

Regional structure–function relationships in mouse aortic valve tissue

Site-specific biomechanical properties of the aortic valve play an important role in native valve function, and alterations in these properties may reflect mechanisms of degeneration and disease. Small animals such as targeted mutagenesis mice provide a powerful approach to model human valve disease pathogenesis; however, physical mechanical testing in small animals is limited by valve tissue size. Aortic valves are comprised of highly organized extracellular matrix compartmentalized in cusp and annulus regions, which have different functions. The objective of this study was to measure regional mechanical properties of mouse aortic valve tissue using a modified micropipette aspiration technique. Aortic valves were isolated from juvenile, adult and aged adult C57BL/6 wild type mice. Tissue tensile stiffness was determined for annulus and cusp regions using a half-space punch model. Stiffness for the annulus region was significantly higher compared to the cusp region at all stages. Further, aged adult valve tissue had decreased stiffness in both the cusp and annulus. Quantitative histochemical analysis revealed a collagen-rich annulus and a proteoglycan-rich cusp at all stages. In aged adult valves, there was proteoglycan infiltration of the annulus hinge, consistent with the observed mechanical differences over time. These findings indicate that valve tissue biomechanical properties vary in wild type mice in a region-specific and age-related manner. The micropipette aspiration technique provides a promising approach for studies of valve structure and function in small animal models, such as transgenic mouse models of valve disease.

Determination of the mechanical properties of DOPC:DOPS liposomes using an image procession algorithm and micropipette-aspiration techniques

Quantification of the mechanical properties of liposomes is critical in helping to predict their behavior during various applications such as targeted drug delivery, response to mechanical characterization or their interactions with isolated cytoskeletal elements. A numerical implementation of the Evans aspiration technique, and an image processing algorithm for measuring deformation of spherical DOPC:DOPS liposomes is presented. Liposomes were aspirated to pressures of −10mmHg (∼−1300Pa). The area expansion and Young's moduli of the liposomes were found to be 0.067Nm−1 (67±4dyn/cm) and 15±1MPa.

Influence of the bending rigidity and the line tension on the mechanical stability of micropipette aspirated vesicles

Micropipette aspiration technique can be very useful for describing the shape transitions of lipid bilayer vesicles with fluid phase coexistence modulated by the line tension and the bending rigidity. For example, composition dependence of the line tension at the liquid-ordered and liquid-disordered phase interface has been investigated using this technique. Recently, using a very simple mechanical model, the instabilities that occur during suction and release experiments of aspirated vesicles have been discussed. Due to its simplicity the model could not predict the influence of the bending rigidity. In this work, we incorporate the contributions from Helfrich bending energy and the line energy in the vesicle free energy and investigate the shape transitions in micropipette aspirated vesicles. We discuss how the critical aspiration pressure depends on the bending moduli of the phases, the line tension at the interface, and the reduced volume of the vesicle. The ratio of the bending moduli that we predict using the critical aspiration pressure data from recent experiments agrees with the literature.

The Constitutive Equation for Membrane Tether Extraction

Membrane tethers or nanotubes play a critical role in a variety of cellular and subcellular processes such as leukocyte rolling and intercellular mass transport. The current constitutive equations that describe the relationship between the pulling force and the tether velocity during tether extraction have serious limitations. In this article, we propose a new phenomenological constitutive equation that captures all known characteristics of nanotube formation, including nonlinearity, nonzero threshold force, and possible negative tether velocity. We used tether extraction from endothelial cells as a prototype to illustrate how to obtain the material constants in the constitutive equation. With the micropipette aspiration technique, we measured tether pulling forces at both positive and negative tether velocities. We also determined the threshold force of 55 pN experimentally for the first time. This new constitutive equation unites two established ones and provides us a unified platform to better understand not only the physiological role of tether extraction during leukocyte rolling and intercellular or intracellular transport, but also the physics of membrane tether growth or retraction.

Aspiration of Biological Viscoelastic Drops

Spherical cellular aggregates are in vitro systems to study the physical and biophysical properties of tissues. We present a novel approach to characterize the mechanical properties of cellular aggregates using a micropipette aspiration technique. We observe an aspiration in two distinct regimes: a fast elastic deformation followed by a viscous flow. We develop a model based on this viscoelastic behavior to deduce the surface tension, viscosity, and elastic modulus. A major result is the increase of the surface tension with the applied force, interpreted as an effect of cellular mechanosensing.

Estimating Young's modulus of zona pellucida by micropipette aspiration in combination with theoretical models of ovum.

The zona pellucida (ZP) is the spherical layer that surrounds the mammalian oocyte. The physical hardness of this layer plays a crucial role in fertilization and is largely unknown because of the lack of appropriate measuring and modelling methods. The aim of this study is to measure the biomechanical properties of the ZP of human/mouse ovum and to test the hypothesis that Young's modulus of the ZP varies with fertilization. Young's moduli of ZP are determined before and after fertilization by using the micropipette aspiration technique, coupled with theoretical models of the oocyte as an elastic incompressible half-space (half-space model), an elastic compressible bilayer (layered model) or an elastic compressible shell (shell model). Comparison of the models shows that incorporation of the layered geometry of the ovum and the compressibility of the ZP in the layered and shell models may provide a means of more accurately characterizing ZP elasticity. Evaluation of results shows that although the results of the models are different, all confirm that the hardening of ZP will increase following fertilization. As can be seen, different choices of models and experimental parameters can affect the interpretation of experimental data and lead to differing mechanical properties.

Validation, In-Depth Analysis, and Modification of the Micropipette Aspiration Technique

The micropipette aspiration technique (MAT) has been successfully applied to many studies in cell adhesion such as leukocyte-endothelium interactions. However, this technique has never been validated experimentally and it has been only employed to impose constant forces. In this study, we validated the force measurement of the MAT with the optical trap and analyzed two technical issues of the MAT, force-transducer offset and cell-micropipette gap, with finite element simulation. We also modified the MAT so that increasing or decreasing forces can be applied. With the modified MAT, we studied tether extraction from endothelial cells by pulling single tethers at increasing velocities and constant force loading rates. Before the onset of tether extraction, an apparently-linear surface protrusion of a few hundred nanometers was observed, which is likely related to membrane receptors pulling on the underlying cytoskeleton. The strength of the modified MAT lies in its capability and consistency to apply a wide range of force loading rates from several piconewtons per second up to thousands of piconewtons per second. With this modification, the MAT becomes more versatile in the study of single molecule and single cell biophysics.

A direct micropipette-based calibration method for atomic force microscope cantilevers.

In this report, we describe a direct method for calibrating atomic force microscope (AFM) cantilevers with the micropipette aspiration technique (MAT). A closely fitting polystyrene bead inside a micropipette is driven by precisely controlled hydrostatic pressures to apply known loads on the sharp tip of AFM cantilevers, thus providing a calibration at the most functionally relevant position. The new method is capable of calibrating cantilevers with spring constants ranging from 0.01 to hundreds of newtons per meter. Under appropriate loading conditions, this new method yields measurement accuracy and precision both within 10%, with higher performance for softer cantilevers. Furthermore, this method may greatly enhance the accuracy and precision of calibration for colloidal probes.

Developmental and osteoarthritic changes in Col6a1‐knockout mice: Biomechanics of type VI collagen in the cartilage pericellular matrix

Chondrocytes, the sole cell type in articular cartilage, maintain the extracellular matrix (ECM) through a homeostatic balance of anabolic and catabolic activities that are influenced by genetic factors, soluble mediators, and biophysical factors such as mechanical stress. Chondrocytes are encapsulated by a narrow tissue region termed the "pericellular matrix" (PCM), which in normal cartilage is defined by the exclusive presence of type VI collagen. Because the PCM completely surrounds each cell, it has been hypothesized that it serves as a filter or transducer for biochemical and/or biomechanical signals from the cartilage ECM. The present study was undertaken to investigate whether lack of type VI collagen may affect the development and biomechanical function of the PCM and alter the mechanical environment of chondrocytes during joint loading. Col6a1(-/-) mice, which lack type VI collagen in their organs, were generated for use in these studies. At ages 1, 3, 6, and 11 months, bone mineral density (BMD) was measured, and osteoarthritic (OA) and developmental changes in the femoral head were evaluated histomorphometrically. Mechanical properties of articular cartilage from the hip joints of 1-month-old Col6a1(-/-), Col6a1(+/-), and Col6a1(+/+) mice were assessed using an electromechanical test system, and mechanical properties of the PCM were measured using the micropipette aspiration technique. In Col6a1(-/-) and Col6a1(+/-) mice the PCM was structurally intact, but exhibited significantly reduced mechanical properties as compared with wild-type controls. With age, Col6a1(-/-) mice showed accelerated development of OA joint degeneration, as well as other musculoskeletal abnormalities such as delayed secondary ossification and reduced BMD. These findings suggest that type VI collagen has an important role in regulating the physiology of the synovial joint and provide indirect evidence that alterations in the mechanical environment of chondrocytes, due to either loss of PCM properties or Col6a1(-/-)-derived joint laxity, can lead to progression of OA.

Adhesion behaviors of human trophoblast cells by contact with endothelial cells

Although it is still not clear whether migratory trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by a mechanism of directional cell division/proliferation, this process involves the attachment and adhesion of trophoblasts to endothelial cells lining the blood vessel walls. This raises the possibility that the cell–cell contact with endothelial cells may regulate trophoblast cell adhesion behaviors according to the surrounding flow condition. To test this, the adhesion forces of early gestation human trophoblast cells (TCs) cultured on glass slides coated with type I rat collagen or cultured with human umbilical vein endothelial cells (HUVECs) were measured quantitatively using a micropipette aspiration technique. Then, the resistance of TCs co-cultured with HUVECs to flow-induced shear stress was assessed with a flow chamber technique. The results showed that the adhesion force of TCs to glass slides coated with collagen was positively correlated with the concentration of collagen. By contact with endothelial cells, the adhesion force and the resistance to shear stress for the TCs were significantly enhanced. The interdiction of integrin β1 interaction remarkably reduced the adhesion forces of TCs to endothelial cells, hence their resistance to shear stress. The results therefore suggest that the contacts of TCs with endothelial cells enhance the adhesion forces of human TCs, partially by regulating with the integrin β1 according to the flow condition (i.e., the shear stress) in such a way to prevent the TCs from being carried downstream by flowing blood.

Viscoelastic Properties of the Aortic Valve Interstitial Cell

There has been growing interest in the mechanobiological function of the aortic valve interstitial cell (AVIC) due to its role in valve tissue homeostasis and remodeling. In a recent study we determined the relation between diastolic loading of the aortic valve (AV) leaflet and the resulting AVIC deformation, which was found to be substantial. However, due to the rapid loading time of the AV leaflets during closure ( approximately 0.05 s), time-dependent effects may play a role in AVIC deformation during physiological function. In the present study, we explored AVIC viscoelastic behavior using the micropipette aspiration technique. We then modeled the resulting time-length data over the 100 s test period using a standard linear solid model, which included Boltzmann superposition. To quantify the degree of creep and stress relaxation during physiological time scales, simulations of micropipette aspiration were preformed with a valve loading time of 0.05 s and a full valve closure time of 0.3 s. The 0.05 s loading simulations suggest that, during valve closure, AVICs act elastically. During diastole, simulations revealed creep (4.65%) and stress relaxation (4.39%) over the 0.3 s physiological time scale. Simulations also indicated that if Boltzmann superposition was not used in parameter estimation, as in much of the micropipette literature, creep and stress relaxation predicted values were nearly doubled (7.92% and 7.35%, respectively). We conclude that while AVIC viscoelastic effects are negligible during valve closure, they likely contribute to the deformation time-history of AVIC deformation during diastole.

Phase Behavior And Domain Structures Of Lipid Membranes Under Tension

The lateral domain structure of lipid membranes is mainly controlled by the thermodynamic characteristics, i.e. composition, temperature and tension. In this study we show experimentally by the combination of fluorescence microscopy and micropipette aspiration techniques and theoretically by phenomenological modeling that the lateral tension of the membrane provides a potent control parameter of the lateral phase behavior and domain structures in lipid membranes. The lateral tension can lead to significant distortions of the phase diagrams and modification of critical behavior, and hence enhancement or suppression of lateral domains.

Nonlinear elastic properties of polyacrylamide gels: Implications for quantification of cellular forces

Because of their tunable mechanical properties, polyacrylamide gels (PAG) are frequently used for studying cell adhesion and migratory responses to extracellular substrate stiffness. Since these responses are known to heavily depend on the tensional balance between cell contractility and substrate mechanical resistance, a precise knowledge of PAG's mechanical properties becomes quite crucial. Using the micropipette aspiration technique, we first exhibited the nonlinear elastic behavior of PAG and then successfully modeled it by an original strain-energy function. This function depends on the Poisson's ratio and on two material parameters, which have been explicitly related to acrylamide and bis-acrylamide concentrations. Implications of these results have been highlighted with regard to traction force microscopy experiments where cellular force quantification is derived from displacements of beads embedded in PAG. We found that considering PAG as a linear elastic medium tends to significantly underestimate traction forces for substrate displacements larger than 2 mum. Interestingly, we also showed that in the range of cellular force amplitude and PAG stiffness currently used in cell traction force experiments, finite size effects become critical for PAG substrate thickness below 60 mum. Thus, our improved characterization of PAG nonlinear mechanical properties through a new constitutive law could have significant impact onto biological experimentations where such extracellular substrates experience large strains.

Viscoelastic behaviour of human mesenchymal stem cells

BackgroundIn this study, we have investigated the viscoelastic behaviour of individual human adult bone marrow-derived mesenchymal stem cells (hMSCs) and the role of F-actin filaments in maintaining these properties, using micropipette aspiration technique together with a standard linear viscoelastic solid model.ResultsUnder a room temperature of 20°C, the instantaneous and equilibrium Young's modulus, E0 and E∞, were found to be 886 ± 289 Pa and 372 ± 125 Pa, respectively, while the apparent viscosity, μ, was 2710 ± 1630 Pa·s. hMSCs treated with cytochalasin D up to 20 μM at 20°C registered significant drop of up to 84% in stiffness and increase of up to 255% in viscosity. At the physiological temperature of 37°C, E0 and E∞ have decreased by 42–66% whereas μ has increased by 95%, compared to the control. Majority of the hMSCs behave as viscoelastic solid with a rapid initial increase in aspiration length and it gradually levels out with time. Three other types of non-typical viscoelastic behavior of hMSCs were also seen.ConclusionhMSCs behave as viscoelastic solid. Its viscoelstic behaviour are dependent on the structural integrity of the F-actin filaments and temperature.

Improvements of anticoagulant activities of silk fibroin films with fucoidan

Fucoidan (FC), an effective anticoagulant constituent extracted from brown algae, was introduced into silk fibroin (SF) for improving its blood compatibility. The SF and SF/FC blend films were characterized by attenuated total reflectance Fourier-transform infrared (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and dynamic contact angle determinator (CA). The in vitro anticoagulant activities of the films were evaluated by activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) measurements. The endothelial cell attachment and proliferation viability on the film were assessed by micropipette aspiration technique and MTT assay, respectively. The testing results indicated that the introduction of FC increased the roughness, hydrophilicity and sulfate component of the film surface without impeding the formation of β-sheet conformation in SF. More important, FC brought excellent anticoagulant activity and better endothelial cell affinity to SF. The SF/FC blend film was hopeful to be used as blood-contacting biomaterials.