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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.

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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.

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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.

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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 kBT 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.

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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.

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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.

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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.

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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.

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The development potential for oocytes can be predicted by their mechanical properties. One important parameter that is measured to calculate oocyte hardness is Cortical Tension (CT). In this work, for the first time, we present the design, simulation, and fabrication of a new aspiration microfluidic chip to measure the CT of oocytes and then predict their maturation capability in the Germinal Vesicle (GV) stage. This high-performance technique facilitates oocyte characterization and is a promising alternative to traditional methods such as MicroPipette Aspiration (MPA). The proposed technique involves considerably simpler operation, less specialized equipment, and less technical skill than MPA. The proposed microfluidic channel also promises faster measurements. It is shown that in order to completely continue the growth process of oocytes in GV stage, the CT should be in a certain range: very low or very high CTs lead to unsuccessful growth. The obtained results show that 79% of oocytes with the CT between 1.5 and 3 nN/µm reach the Metaphase II (MII) stage, whereas the growth for 78% of oocytes with the CT less than 1.5 nN/µm or higher than 3 nN/µm stops at the GV or Germinal Vesicle Break Down (GVBD) stages. Another property, kvis, that points to the viscous behavior of oocytes is also measured. It is seen that 80% of GV oocytes with the kvis values between 15 and 30 k Pa s/m reach the MII stage.

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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.

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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.

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The loss of inner ear hair cells causes permanent hearing and balance deficits in humans and other mammals, but non-mammals recover after supporting cells (SCs) divide and replace hair cells. The proliferative capacity of mammalian SCs declines as exceptionally thick circumferential F-actin bands develop at their adherens junctions. We hypothesized that the reinforced junctions were limiting regenerative responses of mammalian SCs by impeding changes in cell shape and epithelial tension. Using micropipette aspiration and atomic force microscopy, we measured mechanical properties of utricles from mice and chickens. Our data show that the epithelial surface of the mouse utricle stiffens significantly during postnatal maturation. This stiffening correlates with and is dependent on the postnatal accumulation of F-actin and the cross-linker Alpha-Actinin-4 at SC-SC junctions. In chicken utricles, where SCs lack junctional reinforcement, the epithelial surface remains compliant. There, SCs undergo oriented cell divisions and their apical surfaces progressively elongate throughout development, consistent with anisotropic intraepithelial tension. In chicken utricles, inhibition of actomyosin contractility led to drastic SC shape change and epithelial buckling, but neither occurred in mouse utricles. These findings suggest that species differences in the capacity for hair cell regeneration may be attributable in part to the differences in the stiffness and contractility of the actin cytoskeletal elements that reinforce adherens junctions and participate in regulation of the cell cycle.

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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.

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Objective To propose a method for determing cell Poisson’s ratio based on micropipette aspiration technique. Methods Based on the assumption of deformation symmetry, the analytical expression between Poisson’s ratio and the amount of deformation was derived by extracting the extrusion deformation characteristics of the cells under micropipette aspiration according to the generalized Hooke’s law. The accurate determination of Poisson’s ratio of cells was realized according to position changes of markers on the surface of cell membrane. ResultsThe Poisson’s ratio of LNCaP cells in prostate cancer cells was measured. The result showed that the Poisson’s ratio of LNCaP cells was between 0.44 and 0.46, with an average value of 0.45. The influence of the location of the same cell feature points on calculation results of Poisson’s ratio was within the error range of 1.6%. Conclusions This method is simple and feasible, can improve the measurement accuracy of Poisson’s ratio of cells, and is helpful for cell detection and screening by using cell mechanical properties in clinic.

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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.

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BACKGROUND: The interest in mechanics of synthetic and biological vesicles has been continuously growing during the last decades. Liposomes serve as model systems for investigating fundamental membrane processes and properties. More recently, extracellular vesicles (EVs) have been investigated mechanically as well. EVs are widely studied in fundamental and applied sciences, but their material properties remained elusive until recently. Elucidating the mechanical properties of vesicles is essential to unveil the mechanisms behind a variety of biological processes, e.g. budding, vesiculation and cellular uptake mechanisms. SCOPE OF REVIEW: The importance of mechanobiology for studies of vesicles and membranes is discussed, as well as the different available techniques to probe their mechanical properties. In particular, the mechanics of vesicles and membranes as obtained by nanoindentation, micropipette aspiration, optical tweezers, electrodeformation and electroporation experiments is addressed. MAJOR CONCLUSIONS: EVs and liposomes possess an astonishing rich, diverse behavior. To better understand their properties, and for optimization of their applications in nanotechnology, an improved understanding of their mechanical properties is needed. Depending on the size of the vesicles and the specific scientific question, different techniques can be chosen for their mechanical characterization. GENERAL SIGNIFICANCE: Understanding the mechanical properties of vesicles is necessary to gain deeper insight in the fundamental biological mechanisms involved in vesicle generation and cellular uptake. This furthermore facilitates technological applications such as using vesicles as targeted drug delivery vehicles. Liposome studies provide insight into fundamental membrane processes and properties, whereas the role and functioning of EVs in biology and medicine are increasingly elucidated.

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Objective By comparing the physical properties (cell area, volume and elastic modulus) of red blood cells (RBCs) between newborn infants and the elderly over 80 years old, and correlation with the physiological and biochemical parameters such as total cholesterol and glycosylated hemoglobin, the effects of different ages and biochemical parameters on RBC physical properties were analyzed. Methods The mcropipette aspiration was used to measure the surface area, volume and elastic modulus of erythrocytes in newborn infants and the elderly over 80 years old, and the data were analyzed by statistical distribution analysis, correlation analysis and regression analysis. Results The mean values of RBC volume, surface area and elastic modulus in the elderly over 80 years old were smaller than those in newborn infants, and the mean values of RBC mechanical parameters in the same age group were not significantly different. The erythrocytes geometric parameter distribution of newborn infants was more concentrated than that of the elderly, while the elastic modulus distribution of newborn infants was more dispersed than that of the elderly. The mechanical properties of RBCs in newborn infants were highly correlated with the total cholesterol and gestational week; the mechanical properties of RBCs in the elderly were highly correlated with diastolic blood pressure and glycated hemoglobin. Conclusions There are significant differences in physical properties of RBCs between newborn infants and the elderly over 80 years old, and the biochemical parameters that affect physical properties of RBCs at different ages are also different.

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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.

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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.

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Oocysts are the environmentally resistant stage of the protozoan parasite Toxoplasma gondii. They are responsible for foodborne infections in humans and animals worldwide. Infectious oocysts contain sporozoites that have to exit the sporocyst and oocyst walls to initiate replication of the parasite within the host tissues. Given their robustness and resistance to chemical degradation, it is still unclear how the oocyst and sporocyst walls release the sporozoites. This process called excystation is thought to occur in the small intestine as a result of the combined action of digestive agents, yet to be identified. By using an oocyst-macrophage co-culture platform, we previously demonstrated in vitro that the excystation of sporozoites and their differentiation into replicative tachyzoites could occur in absence of digestive factors, following phagocytosis by macrophages. Here, we further characterize the dynamics of the oocyst phagocytosis at the single-cell level by using optical tweezers and micropipette aspiration techniques. Our results show that the oocyst internalization kinetics can vary among a given population of macrophages, but similar processes and dynamics could be observed. Most of the cells manipulate oocysts for ~15 min before internalizing them in typically 30 min. This process mainly involves the actin cytoskeleton of the macrophages. Liberated sporozoites within macrophages then differentiate into tachyzoites within 4-6 h following oocyst-macrophage contact. Tachyzoites appear to develop better in macrophages challenged with free sporocysts or sporozoites than with whole oocysts, suggesting that opening of the oocyst wall is one of the most limiting steps for sporozoite excystation completion.

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