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Extracellular vesicles are promising candidates for novel biomedical applications due to their universal secretion by all organisms. Despite their discovery in 1971, understanding of extracellular vesicles remains in its infancy due to their complex nature and nanoscale dimensions, which make characterization challenging. Extracellular vesicles contain a diverse array of proteins, making them valuable for identifying disease‐specific biomarkers and driving research since 2007. However, identifying these biomarkers remains difficult and expensive. Advancements in extracellular vesicle techniques, including single extracellular vesicle characterization, hold promise for disease diagnosis and personalized medicine. Notably, the biomechanical properties of extracellular vesicles have emerged as a potential diagnosis tool. However, biomechanical characterization has rarely been investigated for disease diagnosis due to limited understanding and a lack of standardized protocols. Recently, significant advancements have been made using various techniques such as atomic force microscopy and micropipette aspiration. This review explores recent developments in biomechanical analysis, demonstrating novel disease diagnostic pathways facilitated by extracellular vesicles and outlining future research directions. [ABSTRACT FROM AUTHOR]

The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles. [ABSTRACT FROM AUTHOR]

Tetraspanin 4, a protein with four transmembrane helices and three connecting loops, senses membrane curvature and localizes to membrane tubes. This enrichment in tubular membranes enhances its diverse interactions. While the transmembrane part of the protein likely contributes to curvature sensitivity, the possible roles of the ectodomains in curvature sensitivity of tetraspanin 4 are still unknown. Here, using micropipette aspiration combined with confocal microscopy and optical tweezers, we show that the extracellular loop 2 contributes to the curvature sensitivity and curvature-induced interactions of tetraspanin 4. To this end, we created truncated tetraspanin 4 mutants by deleting each of the connecting loops. Subsequently, we pulled membrane tubes from giant plasma membrane vesicles containing tetraspanin 4-GFP or its mutants while maintaining controllable membrane tension and curvature. Among the mutations tested, the removal of the extracellular loop 2 had the most significant impact on both the curvature sensitivity and interactions of tetraspanin 4. Based on the results, we suggest that the extracellular loop 2 regulates the affinity of tetraspanin 4 towards curved membranes and affects its lateral interactions.

A sample environment and manipulation tool is presented for single-particle X-ray experiments in an aqueous environment. The system is based on a single water droplet, positioned on a substrate that is structured by a hydrophobic and hydrophilic pattern to stabilize the droplet position. The substrate can support several droplets at a time. Evaporation is prevented by covering the droplet by a thin film of mineral oil. In this windowless fluid which minimizes background signal, single particles can be probed and manipulated by micropipettes, which can easily be inserted and steered in the droplet. Holographic X-ray imaging is shown to be well suited to observe and monitor the pipettes, as well as the droplet surface and the particles. Aspiration and force generation are also enabled based on an application of controlled pressure differences. Experimental challenges are addressed and first results are presented, obtained at two different undulator endstations with nano-focused beams. Finally, the sample environment is discussed in view of future coherent imaging and diffraction experiments with synchrotron radiation and single X-ray free-electron laser pulses.

A sample environment and manipulation tool is presented for single-particle X-ray experiments in an aqueous environment. The system is based on a single water droplet, positioned on a substrate that is structured by a hydrophobic and hydrophilic pattern to stabilize the droplet position. The substrate can support several droplets at a time. Evaporation is prevented by covering the droplet by a thin film of mineral oil. In this windowless fluid which minimizes background signal, single particles can be probed and manipulated by micropipettes, which can easily be inserted and steered in the droplet. Holographic X-ray imaging is shown to be well suited to observe and monitor the pipettes, as well as the droplet surface and the particles. Aspiration and force generation are also enabled based on an application of controlled pressure differences. Experimental challenges are addressed and first results are presented, obtained at two different undulator endstations with nano-focused beams. Finally, the sample environment is discussed in view of future coherent imaging and diffraction experiments with synchrotron radiation and single X-ray free-electron laser pulses. [ABSTRACT FROM AUTHOR]

Cells shield organelles and the cytosol via an active boundary predominantly made of phospholipids and membrane proteins, yet allowing communication between the intracellular and extracellular environment. Micron-sized liposome compartments commonly known as giant unilamellar vesicles (GUVs) are used to model the cell membrane and encapsulate biological materials and processes in a cell-like confinement. In the field of bottom-up synthetic biology, many have utilized GUVs as substrates to study various biological processes such as protein-lipid interactions, cytoskeletal assembly, and dynamics of protein synthesis. Like cells, it is ideal that GUVs are also mechanically durable and able to stay intact when the inner and outer environment changes. As a result, studies have demonstrated approaches to tune the mechanical properties of GUVs by modulating membrane composition and lumenal material property. In this context, there have been many different methods developed to test the mechanical properties of GUVs. In this review, we will survey various perturbation techniques employed to mechanically characterize GUVs.

The success rate in vitro fertilization is significantly linked to the quality of the oocytes. The oocyte's membrane is encapsulated by a shell of gelatinous extracellular matrix, called zona pellucida, which undergoes dynamic changes throughout the reproduction cycle. During the window of highest fertility, the zona pellucida exhibits a softening phase, while it remains rigid during oocyte maturation and again after fertilization. These variations in mechanical properties facilitate or inhibit sperm penetration. Since successful fertilization considerably depends on the state of the zona pellucida, monitoring of the hardening process of the zona pellucida is vital. In this study, we scrutinized two distinct genetic mouse models, namely, fetuin-B wild-type and fetuin-B/ovastacin double deficient with normal and super-soft zona pellucida, respectively. We evaluated the hardening with the help of a microfluidic aspiration-assisted electrical impedance spectroscopy system. An oocyte was trapped by a microhole connected to a microfluidic channel by applying suction pressure. Transient electrical impedance spectra were taken by microelectrodes surrounding the microhole. The time-depending recovery of zona pellucida deflections to equilibrium was used to calculate the Young's modulus and, for the first time, absolute viscosity values. The values were obtained by fitting the curves with an equivalent mechanical circuit consisting of a network of dashpots and springs. The observer-independent electrical readout in combination with a fitting algorithm for the calculation of the viscoelastic properties demonstrates a step toward a more user-friendly and easy-to-use tool for the characterizing and better understanding of the rheological properties of oocytes. [ABSTRACT FROM AUTHOR]

Three constitutive laws, that is the Skalak, neo-Hookean and Yeoh laws, commonly employed for describing the erythrocyte membrane mechanics are theoretically analyzed and numerically investigated to assess their accuracy for capturing erythrocyte deformation characteristics and morphology. Particular emphasis is given to the nonlinear deformation regime, where it is known that the discrepancies between constitutive laws are most prominent. Hence, the experiments of optical tweezers and micropipette aspiration are considered here, for which relationships between the individual shear elastic moduli of the constitutive laws can also be established through analysis of the tension-deformation relationship. All constitutive laws were found to adequately predict the axial and transverse deformations of a red blood cell subjected to stretching with optical tweezers for a constant shear elastic modulus value. As opposed to Skalak law, the neo-Hookean and Yeoh laws replicated the erythrocyte membrane folding, that has been experimentally observed, with the trade-off of sustaining significant area variations. For the micropipette aspiration, the suction pressure-aspiration length relationship could be excellently predicted for a fixed shear elastic modulus value only when Yeoh law was considered. Importantly, the neo-Hookean and Yeoh laws reproduced the membrane wrinkling at suction pressures close to those experimentally measured. None of the constitutive laws suffered from membrane area compressibility in the micropipette aspiration case. [ABSTRACT FROM AUTHOR]

Mechanosensory transduction in Corynebacterium glutamicum plays a major role in glutamate efflux for industrial MSG, whose production depends on the activation of MscCG-type mechanosensitive channels. Dependence of the MscCG channel activation by membrane tension on the membrane lipid content has to date not been functionally characterized. Here, we report the MscCG channel patch clamp recording from liposomes fused with C. glutamicum membrane vesicles as well as from proteoliposomes containing the purified MscCG protein. Our recordings demonstrate that mechanosensitivity of MscCG channels depends significantly on the presence of negatively charged lipids in the proteoliposomes. MscCG channels in liposome preparations fused with native membrane vesicles exhibited the activation threshold similar to the channels recorded from C. glutamicum giant spheroplasts. In comparison, the activation threshold of the MscCG channels reconstituted into azolectin liposomes was higher than the activation threshold of E. coli MscL, which is gated by membrane tension close to the bilayer lytic tension. The spheroplast-like activation threshold was restored when the MscCG channels were reconstituted into liposomes made of E. coli polar lipid extract. In liposomes made of polar lipids mixed with synthetic phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, the activation threshold of MscCG was significantly reduced compared to the activation threshold recorded in azolectin liposomes, which suggests the importance of anionic lipids for the channel mechanosensitivity. Moreover, the micropipette aspiration technique combined with patch fluorometry demonstrated that membranes containing anionic phosphatidylglycerol are softer than membranes containing only polar non-anionic phosphatidylcholine and phosphatidylethanolamine. The difference in mechanosensitivity between C. glutamicum MscCG and canonical MscS of E. coli observed in proteoliposomes explains the evolutionary tuning of the force from lipids sensing in various bacterial membrane environments. [ABSTRACT FROM AUTHOR]

Mechanical stimuli are fundamental in the development of organs and tissues, their growth, regeneration or disease. They influence the biochemical signals produced by the cells, and, consequently, the development and spreading of a disease. Moreover, tumour cells are usually characterized by a decrease in the cell mechanical properties that may be directly linked to their metastatic potential. Thus, recently, the experimental and computational study of cell biomechanics is facing a growing interest. Various experimental approaches have been implemented to describe the passive response of cells; however, cell variability and complex experimental procedures may affect the obtained mechanical properties. For this reason, in-silico computational models have been developed through the years, to overcome such limitations, while proposing valuable tools to understand cell mechanical behaviour. This being the case, we propose a combined continuous-tensegrity finite element (FE) model to analyse the mechanical response of a cell and its subcomponents, observing how every part contributes to the overall mechanical behaviour. We modelled both Atomic Force Microscopy (AFM) indentation and micropipette aspiration techniques, as common mechanical tests for cells and elucidated also the role of cell cytoplasm and cytoskeleton in the global cell mechanical response. [ABSTRACT FROM AUTHOR]

The natural vesicles, microscopic spherical structures defined by a single or many lipid bilayer membranes, not only entrap but are also dispersed in the aqueous environment. The space division between inner and outer compartments is also the basic characteristics of cell membranes playing several essential functions in all living organisms. Thus, vesicles are a simple model system for studying various cellular properties. In the last few decades, synthetic vesicles (or liposomes) have gained substantial popularity from many academia as model membranes and from many pharmaceutical industries as targeted and controlled drug delivery systems. The manufacturing of vesicles with desired characteristics that can entrap and release the drugs as required is one of the major challenges in this research area. To this end, a better understanding of the mechanical and transport properties of vesicles is essential to gain deeper insight into the fundamental biological mechanisms of vesicle formation and cellular uptake. The requirement has brought the modifications in membrane composition (with cholesterol, charged lipid, proteins, peptides, polymers, etc.) and solution conditions (with salts, pH, buffers, etc.). This article mainly focuses on the different techniques developed for studying the mechanical and transport properties of natural/synthetic vesicles. In particular, I thoroughly review the properties such as bending and stretching elastic moduli, lysis tension, and permeability of vesicle membranes. [ABSTRACT FROM AUTHOR]

Mechanosensory transduction in Corynebacterium glutamicum plays a major role in glutamate efflux for industrial MSG, whose production depends on the activation of MscCG-type mechanosensitive channels. Dependence of the MscCG channel activation by membrane tension on the membrane lipid content has to date not been functionally characterized. Here, we report the MscCG channel patch clamp recording from liposomes fused with C. glutamicum membrane vesicles as well as from proteoliposomes containing the purified MscCG protein. Our recordings demonstrate that mechanosensitivity of MscCG channels depends significantly on the presence of negatively charged lipids in the proteoliposomes. MscCG channels in liposome preparations fused with native membrane vesicles exhibited the activation threshold similar to the channels recorded from C. glutamicum giant spheroplasts. In comparison, the activation threshold of the MscCG channels reconstituted into azolectin liposomes was higher than the activation threshold of E. coli MscL, which is gated by membrane tension close to the bilayer lytic tension. The spheroplast-like activation threshold was restored when the MscCG channels were reconstituted into liposomes made of E. coli polar lipid extract. In liposomes made of polar lipids mixed with synthetic phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, the activation threshold of MscCG was significantly reduced compared to the activation threshold recorded in azolectin liposomes, which suggests the importance of anionic lipids for the channel mechanosensitivity. Moreover, the micropipette aspiration technique combined with patch fluorometry demonstrated that membranes containing anionic phosphatidylglycerol are softer than membranes containing only polar non-anionic phosphatidylcholine and phosphatidylethanolamine. The difference in mechanosensitivity between C. glutamicum MscCG and canonical MscS of E. coli observed in proteoliposomes explains the evolutionary tuning of the force from lipids sensing in various bacterial membrane environments.

Cancer alters the structural integrity and morphology of cells. Consequently, the cell function is overshadowed. In this study, the micropipette aspiration process is computationally modeled to predict the mechanical behavior of the colorectal cancer cells. The intended cancer cells are modeled as an incompressible Neo-Hookean visco-hyperelastic material. Also, the micropipette is assumed to be rigid with no deformation. The proposed model is validated with an in-vitro study. To capture the equilibrium and time-dependent behaviors of cells, ramp, and creep tests are respectively performed using the finite element method. Through the simulations, the effects of the micropipette geometry and the aspiration pressure on the colorectal cancer cell lines are investigated. Our findings indicate that, as the inner radius of the micropipette increases, despite the increase in deformation rate and aspirated length, the time to reach the equilibrium state increases. Nevertheless, it is obvious that increasing the tip curvature radius has a small effect on the change of the aspirated length. But, due to the decrease in the stress concentration, it drastically reduces the equilibrium time and increases the deformation rate significantly. Interestingly, our results demonstrate that increasing the aspiration pressure somehow causes the cell stiffening, thereby reducing the upward trend of deformation rate, equilibrium time, and aspirated length. Our findings provide valuable insights for researchers in cell therapy and cancer treatment and can aid in developing more precise microfluidic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The lateral mobility of proteins within cell membranes is usually thought to be dependent on their size and modulated by local heterogeneities of the membrane. Experiments using single-particle tracking on reconstituted membranes demonstrate that protein diffusion is significantly influenced by the interplay of membrane curvature, membrane tension, and protein shape. We find that the curvature-coupled voltage-gated potassium channel (KvAP) undergoes a significant increase in protein mobility under tension, whereas the mobility of the curvature-neutral water channel aquaporin 0 (AQP0) is insensitive to it. Such observations are well explained in terms of an effective friction coefficient of the protein induced by the local membrane deformation.

Tetraspanin 4, a protein with four transmembrane helices and three connecting loops, senses membrane curvature and localizes to membrane tubes. This enrichment in tubular membranes enhances its diverse interactions. While the transmembrane part of the protein likely contributes to curvature sensitivity, the possible roles of the ectodomains in curvature sensitivity of tetraspanin 4 are still unknown. Here, using micropipette aspiration combined with confocal microscopy and optical tweezers, we show that the extracellular loop 2 contributes to the curvature sensitivity and curvature-induced interactions of tetraspanin 4. To this end, we created truncated tetraspanin 4 mutants by deleting each of the connecting loops. Subsequently, we pulled membrane tubes from giant plasma membrane vesicles containing tetraspanin 4-GFP or its mutants while maintaining controllable membrane tension and curvature. Among the mutations tested, the removal of the extracellular loop 2 had the most significant impact on both the curvature sensitivity and interactions of tetraspanin 4. Based on the results, we suggest that the extracellular loop 2 regulates the affinity of tetraspanin 4 towards curved membranes and affects its lateral interactions., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Blood exposure to supraphysiological shear stress within mechanical circulatory support is suspected of reducing red blood cell (RBC) deformability and being primal in the pathogenesis of several secondary complications. No prior works have explored RBC dynamics with the resolution required to determine shear elastic modulus, and/or cell capillary velocity, following exposure to mechanical stresses. Healthy RBCs were exposed to 0, 5, 50, and 100 Pa in a Couette shearing system. For comparison, blood was also exposed to heat treatment—a method that predictably increases RBC rigidity. Shear modulus assessment required aspiration of single RBCs through narrow micropipettes at known suction force. Cell transit velocities were measured within microchannels in regions of fully developed flow. Supraphysiological shear stress increased the elastic shear modulus by 39% and 69% following exposure to 50 and 100Pa, respectively. Cell transit velocity, however, did not change following shear, with concurrent decreases in cell volume likely nullifying increased shear modulus‐friction interactions. Differences observed were consistent with our internal control (heat treatment), supporting that cell mechanics are significantly impaired following supraphysiological‐sublethal shear exposure. Given mechanical circulatory support operates at shear stresses consistent with the present study, it is plausible that these devices induce fundamental impairment to the material properties of RBCs. [ABSTRACT FROM AUTHOR]

A novel facile approach to creating many localized giant polymersomes (GPs) (vesicles) is developed. This method allows for the easy capture and testing of the bulk properties of the GPs. Inkjet printing is shown to produce vesicles of a non‐ionic block copolymer, poly(ethylene oxide)16‐poly(butylene oxide)22 (E16B22). The printer deposits a polymer solution (i.e., water/glucose/polymer) on a hydrophobic glass slide. Upon drying, glucose is left between polymer lamellar. Subsequent hydration of the sample dissolves glucose, and as a result, a concentration gradient is generated, which provides the energy necessary for separating the neutral polymer layers. After about 2 h in water, GPs are observed and are free of organic solvents. This novel methodology provides for the efficient formation of localized GPs. The distribution of the solution allows for a metered approach to releasing the GPs. This combination facilitates the collection of vesicles for micropipette aspiration experiments. [ABSTRACT FROM AUTHOR]

Cytoplasmic viscosity (μc) is a key biomechanical parameter for evaluating the status of cellular cytoskeletons. Previous studies focused on white blood cells, but the data of cytoplasmic viscosity for tumour cells were missing. Tumour cells (H1299, A549 and drug-treated H1299 with compromised cytoskeletons) were aspirated continuously through a micropipette at a pressure of −10 or −5 kPa where aspiration lengths as a function of time were obtained and translated to cytoplasmic viscosity based on a theoretical Newtonian fluid model. Quartile coefficients of dispersion were quantified to evaluate the distributions of cytoplasmic viscosity within the same cell type while neural network-based pattern recognitions were used to classify different cell types based on cytoplasmic viscosity. The single-cell cytoplasmic viscosity with three quartiles and the quartile coefficient of dispersion were quantified as 16.7 Pa s, 42.1 Pa s, 110.3 Pa s and 74% for H1299 cells at −10 kPa (ncell = 652); 144.8 Pa s, 489.8 Pa s, 1390.7 Pa s, and 81% for A549 cells at −10 kPa (ncell = 785); 7.1 Pa s, 13.7 Pa s, 31.5 Pa s, and 63% for CD-treated H1299 cells at −10 kPa (ncell = 651); and 16.9 Pa s, 48.2 Pa s, 150.2 Pa s, and 80% for H1299 cells at −5 kPa (ncell = 600), respectively. Neural network-based pattern recognition produced successful classification rates of 76.7% for H1299 versus A549, 67.0% for H1299 versus drug-treated H1299 and 50.3% for H1299 at −5 and −10 kPa. Variations of cytoplasmic viscosity were observed within the same cell type and among different cell types, suggesting the potential role of cytoplasmic viscosity in cell status evaluation and cell type classification.

Aqueous microgels are distinct entities of soft matter with mechanical signatures that can be different from their macroscopic counterparts due to confinement effects in the preparation, inherently made to consist of more than one domain (Janus particles) or further processing by coating and change in the extent of crosslinking of the core. Motivated by the importance of the mechanical properties of such microgels from a fundamental point, but also related to numerous applications, we provide a perspective on the experimental strategies currently available and emerging tools being explored. Albeit all techniques in principle exploit enforcing stress and observing strain, the realization differs from directly, as, e.g., by atomic force microscope, to less evident in a fluid field combined with imaging by a high-speed camera in high-throughput strategies. Moreover, the accompanying analysis strategies also reflect such differences, and the level of detail that would be preferred for a comprehensive understanding of the microgel mechanical properties are not always implemented. Overall, the perspective is that current technologies have the capacity to provide detailed, nanoscopic mechanical characterization of microgels over an extended size range, to the high-throughput approaches providing distributions over the mechanical signatures, a feature not readily accessible by atomic force microscopy and micropipette aspiration.

Cellular physical properties have been identified to reflect cell states. Existing techniques are able to characterize either mechanical or electrical properties of a cell. This paper presents a micropipette aspiration technique that enables the characterization of both mechanical (instantaneous elastic modulus, equilibrium elastic modulus, and viscosity), and electrical (specific membrane capacitance) properties of the same single cell. Two bladder cancer cell lines (RT4 and T24) with different metastatic potential were used to evaluate the technique. The results showed that high-grade bladder cancer cells (T24, grade III) possess lower viscosity, lower elastic modulus, and larger SMC than the low-grade cancer cells (RT4, grade I). The Naive Bayes classifier was utilized to assess the classification accuracy using single-physical and multi-physical parameters. The classification results confirmed that the use of multi-biophysical parameters resulted in higher accuracy (97.5%), sensitivity (100%), and specificity (95.2%) than the use of a single-physical parameter for distinguishing T24 and RT4 cells. [ABSTRACT FROM AUTHOR]

Among the potential novel therapeutics to treat bacterial infections, antimicrobial peptides (AMPs) are a very promising substitute due to their broad-spectrum activity and rapid bactericidal action. AMPs strongly interact with the bacterial membrane, and the need to have a correct understanding of the interaction between AMPs and lipid bilayers at a molecular level prompted a wealth of experimental and theoretical studies exploiting a variety of AMPs. Here, we studied the effects of magainin H2 (Mag H2), an analog of the well-known magainin 2 (wt Mag 2) AMP endowed with a higher degree of hydrophobicity, on giant unilamellar vesicles (GUVs) concentrating on its permeabilization activity and the effect on the lipid bilayer mechanical properties. We demonstrated that the increased hydrophobicity of Mag H2 affects its selectivity conferring a strong permeabilization activity also on zwitterionic lipid bilayers. Moreover, when lipid mixtures including PG lipids are considered, PG has a protective effect, at variance from wt Mag 2, suggesting that for Mag H2 the monolayer curvature could prevail over the peptide-membrane electrostatic interaction. We then mechanically characterized GUVs by measuring the effect of Mag H2 on the bending constant of lipid bilayers by flickering spectroscopy and, by using micropipette aspiration technique, we followed the steps leading to vesicle permeabilization. We found that Mag H2, notwithstanding its enhanced hydrophobicity, has a pore formation mechanism compatible with the toroidal pore model similar to that of wt Mag 2. [ABSTRACT FROM AUTHOR]

The mechanical properties of living cells are known to be promising biomarkers when investigating the health and functions of the human body. Ionizing irradiation results in vascular injury due to endothelial damage. Thus, the current study objective was to evaluate the influence of continuous radiation doses on the mechanical properties of human umbilical vein endothelial cells (HUVECs), and to identify Young's modulus (E) and viscoelastic behavior. Single-dose (0, 2, 4, 6, and 8 Gy) radiation was applied to HUVECs using a Cobalt-60 treatment machine in the current vitro irradiation study. Thereafter, a micropipette-aspiration technique was used to measure the elastic modulus of the HUVECs in control and radiation-induced samples. Confocal imaging was then performed for following of the cytoskeletal reorganization of the HUVECs in response to the different radiation doses. Significant enhanced adhesion of the elastic modulus of the HUVECs was observed. The dose value was seen to increase from 0 Gy to 8 Gy. A linear relationship was observed between the 0 Gy and 8 Gy doses following an examination of the dose-response curve for elastic modulus after irradiation. The correlation coefficient was found to be 0.955 and the sensitivity of the dose-elastic modulus to be 7.69 Pa..Gy-1 following analysis of the linear portion of the response curve. Also, a significant increment in stiffness accompanied with the considerable drop in creep compliance curve was detected in radiation-induced groups. Biomechanics-based analysis can provide a platform from which to assess the response of the endothelium to radiation when studying vascular system behavior during the cancer therapy process. [ABSTRACT FROM AUTHOR]

Synchronized data from ion currents and optical microscopy have been used to measure the mechanical properties of individual soft microparticles. This new experimental method draws on the signals generated by each particle as it passes through the tip of a pipette. The technique represents an advance on micropipette aspiration (MA), which uses optical microscopy in isolation. The ionic resistance through the tip provides additional information regarding the progress of particle deformation. The augmented technique is demonstrated by studying aspiration of individual bovine erythrocytes into a micropipette. Effective mechanical properties have been deduced by using the ionic current response to measure the dwell time and velocity of erythrocytes. Values obtained for the effective viscosity represent liquid-like deformation of the whole cell, and show a consistent shear thinning effect. The effective elasticity also varies with applied force, and was higher for erythrocytes stored for several weeks. The data show that wall friction is an important factor for the uptake dynamics, and that the erythrocytes were damaged or ruptured when passing through pipettes of opening diameter 0.7 μm, the smallest used. These results demonstrate a new approach to measuring the mechanics of individual bioparticles. • Mechanics of erythrocytes were deduced by measuring ion current during aspiration • Potential cell rupture was identified from current signatures using 0.7 μm pipettes • Apparent viscosity was 4–30 Pa s, with cells exhibiting shear thinning behaviour • In vitro stored cells were found to be stiffer than fresh cells [ABSTRACT FROM AUTHOR]

BACKGROUND: Cancerous transformation of cells affects their mechanical behavior and cytoskeleton structure. OBJECTIVE: The objective of this research is to investigate a correlation between mechanical properties and cytoskeletal structure features in cancer cell formation. METHODS: Micropipette aspiration was used to compare mechanical properties of normal (MCF10A) and cancerous (T47D) epithelial breast cell lines. Immunofluorescence and confocal microscopy were employed for staining and imaging F-actin and microtubules, and quantifying their fluorescent intensity, anisotropy and fiber distribution. RESULTS: Results indicated higher F-actin intensity (43%) and anisotropy (50%) in normal cells compared to cancer cells, although there was no difference in the microtubules intensity between cell lines. Furthermore, reductions of cortex thickness and actin layer index (60%) were observed in suspended cancer cells compared to normal cells. Changes in cell physical properties induced by cancer were attributed to microtubules. The arranged fibrous structure of microtubules in normal cells was replaced by a disorganized structure in cancer cells. Cancerous cells were about four times softer with higher creep compliance compared to normal cells. CONCLUSIONS: Results of this study confirmed that alterations in cell mechanical properties induced by cancer are highly correlated with changes in F-actin and microtubule content and arrangement. It is suggested that such changes can enhance our knowledge of cancer initiation and progression. [ABSTRACT FROM AUTHOR]

Exposure to ionizing radiation is unavoidable for noncancerous cells during the external radiotherapy process. Increasing the dose delivery fraction times leads to increasing the endothelial cell damage. Vascular abnormalities are commonly associated with the alternation of endothelium biomechanical properties. The goal of the present study was to quantify the elastic and viscoelastic properties of human umbilical vein endothelial cells (HUVECs) using the micropipette aspiration technique in conjunction with a theoretical model while an 8 Gy dose was given in four fractions. Confocal imaging was performed for evaluation of cytoskeletal changes during fractionation 60Co radiotherapy. The results indicated an increase in elastic modulus from 29.87 ± 1.04 Pa to 46.69 ± 1.17 Pa while the fractional doses increased from 0 Gy to 8 Gy along with the obvious cytoskeletal changes. Moreover, in the creep behavior of radiated groups, a significant decrease was shown in the time constant and viscoelastic properties. On the other hand, it was observed that the change in the biomechanical properties of the cells while applying a single fraction of 8 Gy was not exactly the same as that in the properties of the radiation-exposed cells while delivering an 8 Gy dose at 2 Gy per fraction. The observed differences in the biomechanical behavior of endothelium provide a quantitative description of radiobiological effects for evaluating the dose-response relationship as a biological dosimetry procedure. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Micro-grooved topography stiffens MSCs and reduces their viscoelasticity. • Substrate stiffness and culture time affects MSCs mechanics, but not interactively. • Substrate topography and stiffness interact to regulate MSCs mechanics. • Substrate topography and culture time interactively modulate MSCs mechanics. Abstract Substrate stiffness and topography are two powerful means by which mesenchymal stem cells (MSCs) activities can be modulated. The effects of substrate stiffness on the MSCs mechanical properties were investigated previously, however, the role of substrate topography in this regard is not yet well understood. Moreover, in vessel wall, these two physical cues act simultaneously to regulate cellular function, hence it is important to investigate their cooperative effects on cellular activity. Herein, we investigated the combined effects of substrate stiffness, substrate topography and culture time on the mechanical behavior of MSCs. The MSCs were cultured on the stiff and soft substrates with or without micro-grooved topography for 10 days and their viscoelastic properties and smooth muscle (SM) gene expression were investigated on days 2, 6 and 10. In general, substrate topography significantly interacted with substrate stiffness as well as culture time in the modulation of cell viscoelastic behavior and SM gene expression. The micro-grooved, stiff substrates resulted in the maximum cell stiffness and gene expression of α-actin and h1-calponin, and these values were detected to be minimum in the smooth, soft substrates. The findings can be helpful in the mechano-regulation of MSCs for vascular tissue engineering applications. [ABSTRACT FROM AUTHOR]

Since the cell was discovered by humans, it has been an important research subject for researchers. The mechanical response of cells to external stimuli and the biomechanical response inside cells are of great significance for maintaining the life activities of cells. These biomechanical behaviors have wide applications in the fields of disease research and micromanipulation. In order to study the mechanical behavior of single cells, various cell mechanics models have been proposed. In addition, the measurement technologies of single cells have been greatly developed. These models, combined with experimental techniques, can effectively explain the biomechanical behavior and reaction mechanism of cells. In this review, we first introduce the basic concept and biomechanical background of cells, then summarize the research progress of internal force models and experimental techniques in the field of cell mechanics and discuss the latest mechanical models and experimental methods. We summarize the application directions of cell mechanics and put forward the future perspectives of a cell mechanics model.

Both Young’s modulus and specific membrane capacitance (SMC) are two important physical parameters for characterizing cell status. In this paper, we utilized a thin-neck-micropipette aspiration system to simultaneously quantify Young’s modulus and SMC value of six types of cell lines in different progression grades, which include four grades from the lowest metastatic potential G1 to the highest potential G4. We investigated how these two physical properties possess heterogeneities in bladder cancer cells with different grades and what roles they might play in grading bladder cancer. The characterization results of these cells of different cancer grades is linearly correlated with the cancer grades, showing that the Young’s modulus is negatively linearly correlated with bladder cancer grades, while SMC shows a positive linear correlation. Furthermore, the combination of these two physical properties on a scatter diagram clearly shows the cell groups with different cancer grades, which means that this combination could be a potential tumor grading marker to identify cancer cells with different metastatic potential.

Fusion of biological membranes is fundamental in various physiological events. The fusion process involves several intermediate stages with energy barriers that are tightly dependent on the mechanical and physical properties of the system, one of which is membrane tension. As previously established, the late stages of fusion, including hemifusion diaphragm and pore expansions, are favored by membrane tension. However, a current understanding of how the energy barrier of earlier fusion stages is affected by membrane tension is lacking. Here, we apply a newly developed experimental approach combining micropipette-aspirated giant unilamellar vesicles and optically trapped membrane-coated beads, revealing that membrane tension inhibits lipid mixing. We show that lipid mixing is 6 times slower under a tension of 0.12 mN/m compared with tension-free membranes. Furthermore, using continuum elastic theory, we calculate the dependence of the hemifusion stalk formation energy on membrane tension and intermembrane distance and find the increase in the corresponding energy barrier to be 1.6 k B T in our setting, which can explain the increase in lipid mixing time delay. Finally, we show that tension can be a significant factor in the stalk energy if the pre-fusion intermembrane distance is on the order of several nanometers, while for membranes that are tightly docked, tension has a negligible effect.

Abstract Many soft biological tissues possess a considerable surface stress, which plays a significant role in their biophysical functions, but most previous methods for characterizing their mechanical properties have neglected the effects of surface stress. In this work, we investigate the micropipette aspiration method to measure the mechanical properties of soft tissues and cells with surface effects. The neo-Hookean constitutive model is adopted to describe the hyperelasticity of the measured biological material, and the surface effect is taken into account by the finite element method. It is found that when the pipette radius or aspiration length is comparable to the elastocapillary length, surface energy may distinctly alter the aspiration response. Generally, both the aspiration length and the bulk normal stress decrease with increasing surface energy, and thus neglecting the surface energy would lead to an overestimation of elastic modulus. Through dimensional analysis and numerical simulations, we provide an explicit relation between the imposed pressure and the aspiration length. This method can be applied to determine the mechanical properties of soft biological tissues and organs, e.g., livers, tumors and embryos. [ABSTRACT FROM AUTHOR]

Cellular mechanical characteristics represent cell ability to produce tissue-specific metabolites. Therefore, to achieve effective cell therapy, a better understanding of the effects of chemo-mechanical stimuli on the mechanical properties of in vitro-treated cells is essential. Herein, we investigated the effects of uniaxial strain on the mechanical properties of mesenchymal stem cells (MSCs) upon transforming growth factor beta 1 (TGF-β1) stimulation. The MSCs were categorized into control and test groups. In one test group, the MSCs were treated by TGF-β1 for 6 d, and in the other, they were additionally subjected to 1-d uniaxial strain on day 2. The cell mechanical properties and smooth muscle (SM) gene expression were assessed on days 2, 4, and 6. During the entire experiment, the MSCs treated by TGF-β1 ± uniaxial strain were induced to differentiate into SM-like cells by significantly upregulation of α-actin, SM22α, and h1-calponin in respect to the control samples. When the MSCs were treated with TGF-β1 alone, their stiffness and viscosity decreased significantly on day 2 and then increased by increase in culture time. When the cells were subjected to 1-d uniaxial strain upon TGF-β1 stimulation, their stiffness and viscosity significantly increased on days 2 and 4 and then decreased on day 6 to a level comparable to that of TGF-β1 group. Different paths were noticeable among the treated samples to reach nearly similar states on day 6. It seems that uniaxial strain activates mechanobiological cascades by which cellular mechanical behavior can be regulated after its removal. However, these effects are transient and would diminish over time. The findings may be helpful in the chemo-mechanical regulation of MSCs. [ABSTRACT FROM AUTHOR]

Radiation therapy has been widely utilized as an effective method to eliminate malignant tumors and cancerous cells. However, subjection of healthy tissues and the related networks of blood vessels adjacent to the tumor area to irradiation is inevitable. The aim of this study was to investigate the consequent effects of fractionation radiotherapy on the mechanical characteristics of human umbilical vein endothelial cells (HUVECs) through alterations in cytoskeleton organization and cell and nucleus morphology. In order to simulate the clinical condition of radiotherapy, the HUVECs were exposed to the specific dose of 2 Gy for 1–4 times among four groups with incremental total dose from 2 Gy up to 8 Gy. Fluorescence staining was performed to label F-actin filaments and nuclei. Micropipette aspiration and standard linear solid model were employed to evaluate the elastic and viscoelastic characteristics of the HUVECs. Radiotherapy significantly increased cell elastic moduli. Due to irradiation, instantaneous and equilibrium Young's modulus were also increased. Radiotherapy diminished HUVECs viscoelastic behavior and shifted their creep compliance curves downward. Furthermore, gamma irradiation elevated the nuclei sizes and to a lesser extent the cells sizes resulting in the accumulation of F-actin filaments within the rest of cell body. Endothelial stiffening correlates with endothelial dysfunction, hence the results may be helpful when the consequent effects of radiotherapy are the focus of concern. [ABSTRACT FROM AUTHOR]

In this study, an aspiration system for elastic spheres was developed to verify the approximate elastic formula for the aspirated length of a single solid-like cell undergoing micropipette aspiration (MPA), which was obtained in our previous study by theoretical analysis and numerical simulation. Using this system, foam silicone rubber spheres with different diameters and mechanical properties were aspirated in a manner similar to the MPA of single cells. Comparisons between the approximate elastic formula and aspiration experiments of spheres indicated that the predictions of the formula agreed with the experimental results. Additionally, combined with the MPA data of rabbit chondrocytes, differences in terms of the elastic parameters derived from the half-space model, incompressible sphere model, and compressible sphere model were explored. The results demonstrated that the parameter ξ (ξ = R/a, where R is the radius of the cell and a is the inner radius of the micropipette) and Poisson’s ratio significantly influenced the determination of the elastic modulus and bulk modulus of the cell. This work developed for the first time an aspiration system of elastic spheres to study the elastic responses of the MPA of a single cell and provided new evidence supporting the use of the approximate elastic formula to determine cellular elastic parameters from the MPA data. [ABSTRACT FROM AUTHOR]

As there are significant variations of cell elasticity among individual cells, measuring the elasticity of batch cells is required for obtaining statistical results of cell elasticity. At present, the micropipette aspiration (MA) technique is the most widely used cell elasticity measurement method. Due to a lack of effective cell storage and delivery methods, the existing manual and robotic MA methods are only capable of measuring a single cell at a time, making the MA of batch cells low efficiency. To address this problem, we developed a robotic MA system capable of storing multiple cells with a feeder micropipette (FM), picking up cells one-by-one to measure their elasticity with a measurement micropipette (MM). This system involved the following key techniques: Maximum permissible tilt angle of MM and FM determination, automated cell adhesion detection and cell adhesion break, and automated cell aspiration. The experimental results demonstrated that our system was able to continuously measure more than 20 cells with a manipulation speed quadrupled in comparison to existing methods. With the batch cell measurement ability, cell elasticity of pig ovum cultured in different environmental conditions was measured to find optimized culturing protocols for oocyte maturation.

The biomechanical properties of single cells show great potential for early disease diagnosis and effective treatments. In this study, a microfluidic device was developed for quantifying the mechanical properties of a single cell. Micropipette aspiration was integrated into a microfluidic device that mimics a classical Wheatstone bridge circuit. This technique allows us not only to effectively alter the flow direction for single-cell trapping, but also to precisely control the pressure exerted on the aspirated cells, analogous to the feature of the Wheatstone bridge that can precisely control bridge voltage and current. By combining the micropipette aspiration technique into the microfluidic device, we can effectively trap the microparticles and Hela cells as well as measure the deformability of cells. The Young’s modulus of Hela cells was evaluated to be 387 ± 77 Pa, which is consistent with previous micropipette aspiration studies. The simplicity, precision, and usability of our device show good potential for biomechanical trials in clinical diagnosis and cell biology research.

Defining the characteristics of cancer stem cells (CSC) has become an important subject in cancer research during the past decade. Although molecular surface expression levels have been used for CSC recognition, the clinical and prognostic impacts of these markers have remained a controversial issue. The finding that cancerous cells are considerably more deformable than normal ones provides the motivation for the hypothesis that the mechanical properties can be used as biomarkers to distinguish between stem-like and non-stem-like cancer cells. In this study, using micropipette aspiration (MA) and intracellular particle tracking (IPT) microrheology, measurements of the whole-cell and local viscoelasticity were made on four breast cancer cell lines with different CSC phenotypes based on their surface markers. Stem-like Hs578T and MDA-MB-231 cell lines were found to be the most deformable, while the non-stem-like MDA-MB-468 line was the least deformable. The non-stem-like BT-20 cell line showed an intermediate deformability. The enhanced deformability for stem-like cells was consistent with the observed lower and more dispersed F-actin content for the stem-like cells. Therefore, the cytoskeleton-related differences in the rheological properties of cancer cells can be a potential biomarker for CSC and eventually lead to novel cancer diagnostic and therapeutic methods. [ABSTRACT FROM AUTHOR]